Riley was born in London in 1931 into a family of printers and moved to Lincolnshire in 1938.

During WWII Riley's father was drafted into the armed services and she was evacuated to a cottage in Cornwall. She was studied at the Royal College of Art (1952–55), where her fellow students included artists Peter Blake, Geoffrey Harcourt and Frank Auerbach. In 1955 Riley graduated with a BA degree.

In 1956, she nursed her father, after he was involved in a serious car crash, and herself suffered a breakdown. After this she worked in a glassware shop and taught children. She eventually joined the J. Walter Thompson advertising agency, as an illustrator, where she worked part-time until 1962. The large Whitechapel Gallery exhibition of Jackson Pollock in 1958, had a major impact on her.

Her early work was figurative with a semi-impressionist style. Between 1958 and 1959 she adopted a style of painting based on the pointillist technique. Around 1960 she began to develop her signature Op Art style. In the summer of 1960 she toured Italy with mentor Maurice de Sausmarez and visited the Venice Biennale with its large exhibition of Futurist works.

In 1961, she acquired her studio in the Vaucluse plateau in France. Back in London, in 1962, Riley was given her first solo exhibition, by Victor Musgrave of Studio One.

In 1968 Riley, with Peter Sedgley and the journalist Peter Townsend, created the artists' organization SPACE (Space Provision Artistic Cultural and Educational), with the goal of providing artists large and affordable studio space.

Riley's mature style, developed during the 1960s, was influenced by a number of sources.

It was during this time that Riley began to paint the black and white works for which she is best known. They present a great variety of geometric forms that produce sensations of movement or colour.

From 1961 to 1964 she worked with the contrast of black and white, occasionally introducing tonal scales of grey. Her paintings have, since 1961, been executed by assistants from her own endlessly edited studies.

Riley began investigating colour in 1967, the year in which she produced her first stripe painting. Following a major retrospective in the early 1970s, Riley began travelling extensively. After a trip to Egypt in the early 1980s, where she was inspired by colourful hieroglyphic decoration, Riley began to explore colour and contrast. In some works, lines of colour are used to create a shimmering effect, while in others the canvas is filled with tessellating patterns.

In many works since this period, Riley has employed others to paint the pieces, while she concentrates on the actual design of her work.

Following a visit to Egypt in 1980–81 Riley created colours in what she called her 'Egyptian palette' and produced works such as the Ka and Ra series, which capture the spirit of the country, ancient and modern, and reflect the colours of the Egyptian landscape. Invoking the sensorial memory of her travels, the paintings produced between 1980 and 1985 exhibit Riley's free reconstruction of the restricted chromatic palette discovered abroad. In 1983 for the first time in fifteen years, Riley returned to Venice to once again study the paintings that form the basis of European colourism. Towards the end of the 1980s Riley's work underwent a dramatic change with the reintroduction of the diagonal in the form of a sequence of parallelograms used to disrupt and animate the vertical stripes that had characterized her previous paintings.