The compulsory purchase order (CPO) was issued for a 345 hectare site in east London, which has some private housing and more than 270 businesses.
Stratford say they are being forced out. The LDA, the mayor's business arm, says it is still prepared to negotiate individual voluntary agreements.
Manny Lewis, chief executive of the LDA, said: "This CPO is a necessary and important step towards the transformation of the Lower Lea Valley and the delivery of the Olympic Park.
Communities 'boosted'
"The regeneration of this area will give local communities the boost they deserve.
"A legacy of 9,000 new homes, double the number of jobs in the Olympic zone area alone, business opportunities, social facilities, including schools, more green space and, of course, fantastic new sports venues.
"The issuing of the CPO does not mean the end of negotiations and, indeed, we would much prefer voluntary agreements with businesses and other land owners."
The majority of those affected will be relocated by the summer of 2007.
One of those affected is Lance Forman who runs a family-owned salmon smoking operation.
He said: "Businesses like our ours are not after compensation. What we want is a site we can operate from that is as good as the one we've got.
"One that give us the same access to the City and West End, so that we can run our businesses successfully like we have done for 100 years.
"What's the point in regenerating it [the area], when you are going to destroy 300 good businesses and 10,000 jobs."
What's the point in regenerating it [the area], when you are going to destroy 300 good businesses and 10,000 jobs 
