Amato was born in Molfetta, Apulia, Italy. He entered the Salesians after completing his novitiate at a Salesian high school. He studied philosophy and theology.
He was ordained a priest on 22 December 1967, becoming a member of the Salesians of Saint John Bosco. He studied at the Salesian Pontifical University gaining a licenitate in philosophy, specializing in Christology. In 1972 he began to teach at the Salesian as an assistant. In 1974 he obtained his doctorate at the Pontifical Gregorian University with a dissertation on The Tridentine pronouncements on the need for sacramental confession in canons 6–9, Session XIV.
In the years 1978 to 1979 he was a fellow of the Ecumenical Patriarchate of Constantinople in Thessaloniki in Greece, at the monastery Orthodox Moní Vlatádon, home of the renowned Institute of patriarchal patristic studies. In 1988 he spent a sabbatical year in Washington, DC, USA, where he began to study the theology of religions.
He was professor of dogmatics at the Salesian Pontifical University, and for twelve years (from 1981 to 1987 and then 1993 to 1997) was dean of the Faculty of Theology.
He served as a consultor to the Congregation for the Doctrine of the Faith and the Pontifical Council for Promoting Christian Unity as well as for the Congregation for Bishops.
His publications include: Trinità in contesto, Biblioteca di Scienze Religiose 110 (Roma: LAS, 1994); La Catechesi al traguardo. Studi sul Catechismo della Chiesa Cattolica, a cura di Angelo Amato, Enrico dal Covolo e Achille M. Triacca, Biblioteca di Scienze Religiose 127 (Roma: LAS, 1997); Il vangelo del Padre (Bologna: EDB, 1998); Gesù il Signore. Saggio di cristologia, Corso di Teologia Sistematica 4 (Bologna: EDB, 1999); Maria e la Trinità. Spiritualità mariana ed esistenza cristiana – Alma Mater (Roma: San Paolo Edizioni, 2000); Il celibato di Gesù (Città del Vaticano: Libreria Editrice Vaticana, 2010); I santi della chiesa (Città del Vaticano: Libreria Editrice Vaticana, 2010).
On 19 December 2002, Pope John Paul II appointed him as Secretary of the Congregation for the Doctrine of the Faith and titular archbishop of Sila. He was consecrated bishop on 6 January 2003 by Pope John Paul II with archbishops Leonardo Sandri and Antonio Maria Vegliò serving as co-consecrators.
In addition to his role as CDF Secretary, Amato served as a consultor to the Pontifical Councils for Christian Unity and Interreligious Dialogue.
In an April 2007 address to chaplains, he denounced same-sex marriage and abortion and criticized the Italian media's coverage of them, saying that they are evils "that remain almost invisible" due to media presentation of them as an "expression of human progress."
The belief that the Catholic Church persecuted Galileo Galilei for pointing out that the Earth goes round the Sun was quite wrong, the Secretary of the Vatican's Doctrinal Congregation, Archbishop Amato claimed. Citing a letter recently discovered in the Vatican's archive, Archbishop Amato said it proved the church had treated him very well.
The letter, sent by the Commissioner of the Holy Office to Francesco Barberini in 1633, expressed the Pope's concern that the trial of the scientist accused of heresy be concluded quickly as his health was poor. Archbishop Amato told the Italian weekly magazine Famiglia Cristiana that the letter proved that the church's attitude to the great astronomer was benign.
After the 2005 Papal conclave, Amato was the first person received in private audience by the new Pope Benedict XVI, who until his election had been Prefect of the Congregation of which Amato continued to be Secretary until 9 July 2008, when Pope Benedict named him Prefect of the Congregation for the Causes of Saints. Thus, Amato oversees the process which leads to the canonisation of saints, which includes preparing a case, including the approval of certified miracles. The case is presented to the pope, who decides whether or not to proceed with beatification or canonisation. Amato is the second CDF Secretary to lead the Causes of Saints dicastery, the first being Alberto Bovone.
On 6 July 2010 he was appointed a member of the Congregation for Divine Worship and the Discipline of the Sacraments. On 16 October 2010 he was appointed by Pope Benedict as a member of the Congregation for the Doctrine of the Faith for a five-year renewable term. It will be renewed until his 80th birthday. Being resident in Rome, he is invited to attend not only the plenary meetings of those departments, which in principle are held every year, but also the ordinary meetings.
Most recently, he beatified a Japanese Servant of God, Dom Justo Takayama who died in the land of the Philippines and was buried there. He was the first daimyo who was buried in the Philippines. He beatifued him (Ukon Takayama) on 7 February 2017 in Japan which is the homeland of the latter blessed.
Amato was created and proclaimed Cardinal-Deacon of S. Maria in Aquiro by Pope Benedict XVI in the consistory of 20 November 2010. On 29 December 2010 Cardinal Amato was appointed member of the Pontifical Council for Promoting Christian Unity.
On Friday 14 January 2011, Pope Benedict XVI signed a decree attributing a miracle to the intercession of the Venerable Servant of God, Pope John Paul II, clearing the way for his beatification, which took place on 1 May 2011. Cardinal Amato said that the "Papal dispensation of the 5-year waiting period before opening a cause, and the second was the placing of the cause on a 'fast track' that by-passed the waiting list. There were, however, no corners cut with regard to the rigor and accuracy of procedure. The case was treated like any other, following all the steps prescribed by the law of the Congregation for the Causes of Saints. On the contrary, if I may speak further to one of my first observations: precisely in order to honor the dignity and the memory of this great Pope, to avoid any doubt and overcome any difficulties, the case was subjected to particularly careful scrutiny."
Amato was considered papabile for the papal conclave of 2013 but it was Jorge Mario Bergoglio who emerged as Pope Francis. In June 2013 Cardinal Amato reached the age of 75 and therefore submitted his age induced resignation to the pope.
A miracle attributed to the intercession of the Paul VI was discovered, voted on, and was approved on 9 May 2014 by Pope Francis in an audience with Cardinal Amato. The beatification ceremony for Paul VI was held on 19 October 2014, which means that the late pope will be called "Blessed".