In 1597 his earliest known work, the Cittarn Schoole, was published in London; it contains 58 of his own compositions, and is considered to be one of the most important sources of music for cittern.

Two years later, in 1599, he published a collection of consort music, Pavans, Galliards, Almains, and other short Aeirs both grave, and light, in five parts, for Viols, Violins or other Musicall Winde Instruments, consisting of 65 different compositions.

It is from the title pages of both of these publications that we learn that he was a 'Gentleman and Servant to her most excellent Maiesti'" and that he in some fashion served Queen Elizabeth I, although it is uncertain what his exact duties were.

He does not appear in any of the registers or accounts of the Chapel Royal. In January of 1599 he travelled as a letter courier 'for her Maiesties service.' According to a letter written by his wife, he suffered from a bad cold in November,1602, which was the cause of his death at the end of that month.

Holborne's literary interests are hidden in his instrumental works, and specifically in their epigrammatic and mystifying titles. Little by little it is being discovered that each fanciful title connects with specific poetry or prose.

Undoubtedly, however, Holborne's most important work is the 1599 collection of instrumental pieces; it is one of the early prints of English consort music, the first of which appeared in 1590.

Printed and manuscript collections of ensemble music became more plentiful particularly during the reign of James I, who succeeded Elizabeth I on her death in 1603, and the fantasy took ever greater precedence over the dances.