His father was the lexicographer, James Knowles (1759-1840), cousin german of Richard Brinsley Sheridan. The family removed to London in 1793, and at the age of fourteen Knowles published a ballad entitled The Welsh Harper, which, set to music, was very popular. The boy's talents secured him the friendship of Hazlitt, who introduced him to Lamb and Coleridge. He served for some time in the Wiltshire and afterwards in the Tower Hamlets militia, leaving the service to become pupil of Dr Robert Willan (1757-1812). He obtained the degree of M.D., and was appointed vaccinator to the Jennerian Society.
Although, however, Dr Willan generously offered him a share in his practice, he resolved to forsake medicine for the stage, making his first appearance probably at Bath, and playing Hamlet at the Crow Theatre, Dublin. At Wexford he married, in October 1809, Maria Charteris, an actress from the Edinburgh Theatre.
In 1810 he wrote Leo, in which Edmund Kean acted with great success; another play, Brian Boroihme, written for the Belfast Theatre in the next year, also drew crowded houses, but his earnings were so small that he was obliged to become assistant to his father at the Belfast Academical Institution. In 1817 he removed from Belfast to Glasgow, where, besides conducting a flourishing school, he continued to write for the stage.
His first important success was Caius Gracchus, produced at Belfast ~fl 1815; and his Virginius, written for Edmund Kean, was first performed in 1820 at Covent Garden. In William Tell (1825) Macready found one of his favorite parts. His best-known play, The Hunchback, was produced at Covent Garden in 1832. The Wife was brought out at the same theatre in 1833; and The Love Chase in 1837.
In his later years he forsook the stage for the pulpit, and as a Baptist preacher attracted large audiences at Exeter Hall and elsewhere. He published two polemical works: the Rock of Rome and the Idol Demolished by its own Priests in both of which he combated the special doctrines of the Roman Catholic Church. Knowles was for some years in the receipt of an annual pension of £200, bestowed by Sir Robert Peel. He died at Torquay on the 30th of November 1862.
A full list of the works of Knowles and of the various notices of him will be found in the Life (1872), privately printed by his son, Richard Brinsley Knowles (1820-1882), who was well known as a journalist.