New walls, flood gates and other measures are now in place along a five-mile stretch of the River Don, stretching from the city centre by the Wicker to the M1 near Meadowhall.
The river has also been dredged of the debris which contributed to flooding in the past.
The work to protect some 500 companies, safeguarding 5,000 jobs and spurring new investment in the industrial heartlands of the Lower Don Valley, is the first major defence project to be completed in the city since the 2007 flood killed two people and devastated homes and businesses.
It is the first time companies in the country have contributed to the costs of flood protection through the creation of a Business Improvement District.
Between them, those firms provided £1.4m towards the £20.7m cost, of which the Environment Agency and Defra (Department for Environment, Food and Rural Affairs) funded more than 90 per cent.
The project, which is one of the last to have been completed by doomed government contractor Carillion, is designed to protect the valley from flooding so severe it should only happen once every 100 years. Previously, the land was only protected against a one in 25 year flood.
Speaking at today's unveiling, Richard Wright, executive director of Sheffield Chamber of Commerce, said: "Thanks to the defences, we've already been able to help one business, which had been offered insurance costing five times what it was paying. reduce that charge to one-and-a-half times the existing rate.
"We're also working with two other businesses, who are getting really favourable replies from insurers. You're starting to see real benefits."
This is the first of six major flood defence projects planned for the city, with remaining schemes to protect homes and businesses in areas including the Upper Don, Sheaf and Porter valleys estimated to cost a further £93m.
Sheffield Council is still in talks to secure funding for that work, which it claims would enable 27,000 new homes to be built and 15,000 new jobs to be created.
It intends to publish more detailed proposals soon and begin work towards the end of the year or early in 2019, with a phased approach so individual elements can be delivered as money is secured.
Councillor Bryan Lodge, the council's cabinet member for environment, said: "I think everyone should be very proud of what's been achieved along the Lower Don Valley. It's given existing businesses increased confidence and we're already seeing new investment.
"This shows what can be achieved, and we're not prepared to wait to protect people, businesses and homes elsewhere. We want to get out there and provide added protection in stages as the funding becomes available."
Clive Betts, MP for Sheffield South East, claimed the outstanding flood defence projects could free up much-needed space for new homes and businesses in the city, preventing the need to build on greenfield land.
"The Government needs to recognise the enormous benefits of spending a bit more money on flood defences, which will help grow the local economy and create new jobs and housing," he said.
The project took seven years from conception to completion and was not always straightforward, with an old bomb and a body being pulled from the river, and work even being paused at one stage to protect otters in the river.
Forgemasters and Meadowhall shopping centre were two of the worst-hit businesses when the valley flooded in 2007, along with Gripple, whose Savile Street factory ended up under five feet of water
Gordon Macrae, Gripple's special projects manager, recalled wading waist-high into the water back then, secured by one of the firm's own wire fasteners, to rescue people who were stranded.
He said the company never considered moving in the wake of the disaster, as it was 'passionate' about manufacturing in Sheffield, but the new defences gave it added confidence to continue its growth.
"It's an amazing scheme because it gives businesses like ours the resilience to be able to continue to invest," he said.
While many firms were left counting the cost of the 2007 flood, Gripple turned adversity to its advantage, spotting the opportunity to expand its range and develop flood defence products.
Lower Don Valley Flood Defence Project in numbers
500 - the number of businesses safeguarded
5,000 - the number of existing jobs protected
700 - the length in metres of new and raised walls along Meadowhead Road
5 - the length in miles along which defences have been bolstered
2 - the maximum height of new walls built
£1.4bn - the total being contributed by 260 businesses over five years