The collection of about 4,000 works has been without a permanent home since the original Guildhall Art Gallery was destroyed by fire during the Second World War.
But now, the Corporation, Britain's biggest sponsor of the arts after the Government and the BBC, has built a gallery on the same site as its predecessor in Victorian chambers beneath the Old Print Room of the Guildhall. Around 250 works will go on display at any one time, and the gallery will be open seven days a week.
Anthony Moss, chairman of the Libraries and Guildhall Art Gallery Committee, said the original gallery was set up in 1886 with the intention of "supporting, encouraging and developing a collection of art worthy of the capital city".
He said: "The sentiment is true today. We believe there should be an art gallery in the heart of the City of London that the 250,000 city workers and other visitors can enjoy." The collection has works dating back to the 16th century, and by painters such as Constable, Millais and Rossetti.
Archaeological investigation of the site began in 1987, and uncovered medieval and Saxon remains, and part of a Roman amphitheatre. Its existence had been suspected but its location was unknown until the pounds 70m excavation and construction project began.