The remains of the Curtain Theatre, which opened in 1577, were found behind a pub in Shoreditch, east London, as part of regeneration works.
The venue was immortalized as “this wooden O” in the prologue to Henry V.
It is hoped the site could be opened to the public, with plays staged there in the future.
Archaeologists from the Museum of London Archaeology (MOLA) stumbled across parts of the playhouse’s yard and gallery walls after development began on the site last October.
“This is a fantastic site which gives us unique insight into early Shakespearean theatres,” lead archaeologist Chris Thomas said.
The Curtain Theatre was operated by theatre manager James Burbage and was home to Shakespeare’s Company, the Lord Chamberlain’s Men, from 1597 until The Globe opened two years later.
The theatre disappeared from historical records in 1622 but could have remained in use until the outbreak of the Civil War, 20 years later.
Plays thought to have premiered there include Henry V, Romeo and Juliet and Ben Jonson’s Every Man in His Humour.
“This is one of the most significant Shakespearean discoveries of recent years,” a spokesman for Plough Yard Developments, which owns the site, said.
“Although The Curtain was known to have been in the area, its exact location was a mystery.
“The quality of the remains found is remarkable and we are looking forward to working with MOLA, [the] local community and Shakespearean experts to develop plans that will give the public access to the theatre remains as part of a new development.”
Royal Shakespeare Company artistic director Michael Boyd added: “I look forward to touching the mud and stone, if not wood, and feeling the presence of that space where Shakespeare’s early work, including the histories, made such a lasting impact.”
Further excavations are expected to take place later this year.
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