Middleton was educated in Norwich and studied art under the landscape painters John Berney Ladbrooke and Henry Bright.

Middleton's death in 1856 from tuberculosis, which cut short his career at the age of 29, has been described by the art historian Josephine Walpole as "the supreme tragedy for the Norwich School of painters".

[17][note 1] Little of his early childhood has been documented and his biographers do not mention any siblings, although the Middletons' memorial in St Stephen's Church includes an unnamed daughter who died in infancy.

In addition to running a glass colouring business, Middleton senior undertook plumbing and painting work, and studied and collected ferns in his spare time.

[13] Ladbrooke, a Norwich School artist who produced a large output of works despite having to earn a living as a drawing master,[26] particularly influenced Middleton's oil technique.

[29] Following his father's death in April 1848,[note 4] Middleton moved back to Norwich in order to take over the running of the family business.

[22] He exhibited in London from 1847 to 1855, despite the interruption caused by his business commitments: he showed fourteen pictures at the Royal Academy and fifteen at the British Institution, where his work was acclaimed.

[36] According to the author and art historian William Frederick Dickes, Middleton was the devoted young friend of Lound,[27] who probably introduced him to the works of Thirtle, as well as to photography.

[37] Lound worked as a brewer clerk and an insurance agent, whose wealth allowed him to pursue his enthusiasm for collecting, copying and producing art.

[40] Along with Middleton, Bright and Leman, Lound was a member of the Committee of the Norfolk and Norwich Association for the Promotion of the Fine Arts (1848–1852).

[43] Middleton befriended William Johnson Jennis Bolding, a resident landowner and farmer in the coastal village of Weybourne.

Middleton was responsible for enrolling him as a member of the Norwich Photographic Society, which held exhibitions that included William Bolding's work.

[44] A small etching by John Middleton, made en plein air, closely resembles a photograph by Bolding, showing that the friends were working together.

[21] According to the art historian Francis Cheetham, Lane over the Hill shows Bright's capacity to "use watercolour with (the) freshness and simplicity which characterises the finest work of John Middleton".

[49] Their friendship was not always smooth: in a letter of 22 February 1850 to Thomas Lound's son, Bright wrote, "J. Middleton dined here tonight, he was wondrous polite.

[52] His will, which was proved in Norwich in July 1857, stipulated that Mary Middleton should inherit both the family business and the remaining funds from the estate.

[54] According to Hemingway, Middleton's death in 1856 marked a decline in the quality of painting produced in the region, and he was the last significant watercolourist to be based in East Anglia.

An idea of his artistic output can be obtained from the catalogue of a sale of his works that took place in June 1883, following the death of Mary Middleton earlier that year.

Middleton produced works using clear colours and incisively-outlined forms, boldly using the white of his paper and being, in Clifford's words, "delighted in the potentialities of his medium".

[58] The author Robert Brall considers Middleton's best paintings to have an "astonishingly modern" look, in comparison with the elaborate detail found in contemporary works.

[54][62] His etchings required a different technique from his watercolours: their delicacy and intricacy are said by the author Geoffrey Searle to resemble the work of John Crome.

[11] In contrast to his watercolours, his etchings are mostly depictions of wooded landscapes, ranging in tone from those that are heavily inked, to others with a light, silvery touch and delicate lines.

[69] His landscape photographs are reminiscent in their contrasting dark and light tones of the large paintings produced by Bright in the late 1840s, such as Study of a Beech Tree.