The Panamanian President, Juan Carlos Varela, described the waterway as a route that would unite the world.
The president thanked the nearly 30,000 people who had worked on the canal`s expansion.
Construction on the new lane for the canal, which runs for 77km (48 miles), began in 2007 and was due to finish in 2014.
But strikes and disputes over costs delayed the $5.2bn (£3.8bn) project.
Panama Canal expansion prompts safety concerns
The original Panama Canal was first used in August 1914. It was built by the US and handed over to local control in 1999.
The expansion allows a new, much-larger generation of container ships to pass through the isthmus.
Some 35 to 40 vessels transit the waterway everyday.
Panama hopes the expansion will increase the revenues it gets from the canal, reported to have been $2.6bn (£1.9bn) in 2015.
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