The Duke of Cambridge arrived at London Bridge Station on a new Class 700 Thameslink train where he was greeted by transport secretary Chris Grayling MP and Network Rail boss Mark Carne.
Once on the new ground-level station concourse, the Prince met groups of apprentices, project leaders and staff involved in the redevelopment.
They were then given a song performance by children from a local primary school.
Guests at the ceremony included Southwark Council leader Peter John, the Bishop of Southwark Rt Revd Christopher Chessun and the Dean of Southwark Very Revd Andrew Nunn.
The dean presented Prince William with a copy of the children's book about Southwark's cathedral cat for princes George and Louis and Princess Charlotte.
"Today, we are indebted to the thousands of men and women who have built a modern, accessible and iconic station fit for London's future, and the patience of passengers who will now see a transformation in their journeys," said transport secretary Chris Grayling MP.
"Thanks to government-sponsored investment, London Bridge Station will be the beating heart of the Thameslink, offering thousands of commuters more frequent and reliable services, with modern new trains using pioneering world-first technology to enable better journeys across the entire route.
"It is my honour to welcome the Duke of Cambridge to London Bridge station to officially declare it open."
Mark Carne, chief executive of Network Rail, said: "This station has been rebuilt from its Victorian foundations upwards by a team of engineers while still providing a service for the 48 million people who use the station every year to deliver it on the very day we said we would five years ago.
"This is a station that represents a transformation in passenger experience, a catalyst for economic growth and a world first in the use of Digital Railway technology demonstrating our vision of the future.
"I give my thanks to the great people and great teams behind this fantastic project, as well as to our customers for their patience and understanding during these major works."
London Bridge station is the oldest station in central London and was originally built in 1836. Today it caters for around 48 million passengers per year, making it one of the busiest stations in the country.
There are still a few jobs to finish at the rebuilt station, including the completion of the new north-south pedestrian route along Stainer Street, parallel with the new concourse.
A major timetable revision begins on Sunday 20 May, with the full-time return of cross-London Thameslink services to London Bridge for the first time since January 2015.
The Thameslink network is also gaining new destinations including Peterborough and Cambridge to the north of London, and Rainham and Horsham south of the river.
• October 2010: Boris Johnson lobbies Government not to cut station rebuild plans
• November 2010: Government confirms station will be rebuilt by 2018
• April 2011: designs for the new station are published
• May 2011: new Borough Viaduct slid into place
• July 2011: planning application submitted
• December 2011: new station wins backing of Southwark planning committee
• May 2012: preparatory works begin
• December 2012: campaigners mourn end of London Bridge to Victoria link
• February 2013: Roman remains found below the station
• August 2013: nine-day part-closure of station
• January 2014: first new platforms take shape
• August 2015: Network Rail fined £2 million
• August 2016: first section of new concourse opens
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