Herluf Trolle was born at Lillö, Scania into the high noble Trolle line of Swedish-origin. He was son of Kirsten Herlufsdatter Skave and Sir Joachim Arvidsen Trolle, Lord of Lilloe; grandson of justiciar Arvid Trolle, Lord of Bergkvara, and the latter's second wife Beate Iversdatter of the Thott, heiress of Lilloe and daughter of lord Iver Axelsen of the Thott, fiefholder of the island of Gulland.
At the age of nineteen Trolle went to Vor Frue Skole at Copenhagen, subsequently completing his studies at Wittenberg University from 1536-1537. Here he adopted the views of Philipp Melanchthon, with whom he was in intimate correspondence for some years.
His marriage with Birgitte, the daughter of Lord High Treasurer Mogens Gøye, brought him a rich inheritance, and in 1557 he was summoned to the membership of the High Council of Denmark. Both Christian III and Frederik II had a very high opinion of Trolle's trustworthiness and ability and employed him in various diplomatic missions. Herluf Trolle was, indeed, richly endowed by nature, and his handsome face and lively manners made him popular everywhere. His one enemy was his wife's nephew Peder Oxe, the subsequently distinguished finance minister, whose narrow grasping ways, especially as the two men were near neighbors, did not contribute towards family harmony. It was Trolle whom Frederik II appointed to investigate the charges of malversation brought against Oxe. Both Trolle and his wife were far renowned for their piety and good works, and their whole household had to conform to their example or seek service elsewhere.
A man of culture, moreover, he translated David's 31st Psalm into Danish verse. He also promoted literature and learning by educating poor students both at home and abroad, endowing Latin schools and encouraging historical research.
In 1559, Trolle was appointed Admiral and Inspector of the Fleet, a task which occupied all his time and energy. In 1563 he superseded the aged Peder Skram as admiral in chief in the Northern Seven Years' War. On 20 May he went to sea with twenty-one ships of the line and five smaller vessels and, after uniting with a Lübeck squadron of six line ships, encountered, off the isle of Öland, a superior Swedish fleet of thirty-eight ships under Jacob Bagge. Supported by two other Danish ships, Trolle attacked the Swedish flagship Mars (also known as Makalös - "Matchless" or Jutehataren, "Hater of Jutes"), then the largest warship in northern waters, but was beaten off at nightfall. The fight was renewed at six o'clock the following morning, when the Makalös was again attacked and forced to surrender, but blew up immediately afterwards, no fewer than 300 Lübeck and Danish sailors perishing with her. But the Swedish admiral was captured and the remnant of the Swedish fleet took refuge at Stockholm.
Despite the damage done to his own fleet and flagship Fortuna by this great victory, Trolle, on 14 August, fought another but indecisive action with a second Swedish fleet under the famous Finnish admiral Klaus Horn, Lord of Joensuu, his distant kinsman, and kept the sea until 13 October. Trolle spent the winter partly at his castle of Herlufsholm completing his long-cherished plan of establishing a school for all classes, and partly at Copenhagen equipping a new fleet for the ensuing campaign. On 1 June 1565 he set sail with twenty-eight liners, which were reinforced off Fehmarn by five Lübeck vessels. Horn had put to sea still earlier with a superior fleet and the two admirals encountered off Fehmarn on 4 June. The fight was severe but indecisive, and both commanders finally separated to repair their ships. Trolle had been severely wounded in the thigh and shoulder, but he would not let the ship's surgeon see to his injuries until every one else had been attended to. This characteristic act of unselfishness was his undoing, for he died at Copenhagen on 25 June, seventeen days after they had put him ashore.
He was buried at Herlufsholm together with his wife Birgitte Gøye in a tomb made by the Flemish sculptor Cornelis Floris.