Lobley's Bridge, known as the "bridge to nowhere", was constructed in 1973 as part of a proposed bypass around the city which was never built.
Developments have now been put up on either side of the motorway and the bridge will link the two.
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Lobley's Bridge has been used over the years as a footpath, and as a car park for a nearby nature reserve.
The county's development co-ordination and transport planning manager, John Seddon, said if it was built today it would cost about £1.5m.
"When the M5 was built, there was a plan to build a relief road on the south side of Gloucester, that's why this bridge was built," he said.
"We're now ready to open it to traffic and a bus service will come into service on Monday, giving a direct link from Brockworth into the city centre."
(NS/JM)
