Mr Obama said: "I don't want to pretend that today marks the end of our economic problems, nor does it constitute all of what we're going to have to do to turn our economy around, but today does mark the beginning of the end."
He said part of the plan would put Americans back to work in critical areas such as roads and rail infrastructure.
"[Today is] the beginning of first steps to set our economy on a firmer foundation," Mr Obama told an audience at a museum in Denver.
Mr Obama also hopes to stimulate technical innovation.
"Just as President Kennedy sparked an explosion of innovation when he set America's sights on the Moon, I hope this investment will ignite our imagination once more, spurring new discoveries and breakthroughs in science, in medicine, in energy, to make our economy stronger and our nation more secure, and our planet safer for our children."
US Vice-president Joe Biden praised Mr Obama's swift work since coming to office in January.
"Less than a month into his presidency, the president is about to sign into law what is, I believe, a landmark achievement.
"Because of what he did America can take a first very strong step leading us out of this very difficult road to recovery we find ourselves with. So, on behalf of our country and its people, Mr President, let me presume to say: thank you, we owe you a great deal."
$240bn in tax breaks for individuals and businesses
The plan got no Republican support in the House of Representatives and just three Republican votes in the Senate.
As the bill was signed, the Republican National Committee chairman Michael Steele said Americans looking for jobs would be disappointed by it.
He dismissed the president's claims of a bi-partisan approach.
"In these difficult economic times, it is imperative that Republicans and Democrats work together to create new jobs and grow the economy.
"Instead, Congressional Democrats worked behind closed doors to write legislation that will fall short of creating the promised new jobs, but will guarantee a larger debt burden on our children and grandchildren."
The bill also includes a controversial "Buy American" provision that, despite being watered down, has angered US trading partners.
On Monday, Brazil's Foreign Minister, Celso Amorim, threatened to challenge the legality of the clause at the World Trade Organization (WTO).
"It's a complex legal analysis, but we're doing it," Mr Amorim told state television. "[Going to the WTO] is a real option," he added.
The approved plan stipulates that public works and building projects funded by the stimulus use only US-made goods, including iron and steel.
The EU and Canada had earlier said that provisions favouring American-produced materials for government projects risked provoking retaliatory protectionist measures.
Earlier this month, Brazil's President Luiz Inacio Lula da Silva told the BBC that in the good years, the rich countries had talked a lot about free trade and the market.
Now they had created a crisis they should not turn to the protectionism which had so often held the world back, President Lula said.
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