Morales has said that the changes will "re-found Bolivia," South America's poorest country, and end a political and social order inherited from Spanish colonial times.
"This is the second independence, the true liberation of Bolivia," Morales said upon signing the charter.
The changes to the constitution won the approval of 61 per cent of voters in a referendum held on January 25.
But the proposals were rejected in Santa Cruz, Tarija, Beni and Pando, wealthy regions, which hold much of the country's natural resources and where anti-Morales sentiment runs deep.
"In the name of the excluded, this constitution excludes another part of the country," Jorge Lazarte, and opposition politician, said.
"That doesn't guarantee its permanence."
Morales denied the charges and told his opponents: "You can take me from the presidential palace, you can kill me, (but) the mission has been accomplished for the refounding of Bolivia".
Guests at the ceremony in El Alto included Jose Miguel Insulza, the head of the Organisation of American States, and Rigoberta Menchu, the indigenous Guatemalan activist, Nicolas Maduro, the foreign minister of Venezuela, and 1992 Nobel Peace prize winner.
Although Morales is widely popular, his rise and the constitutional changes have heightened divisions in the country, which erupted in violence in September when 20 indigenous government supporters were killed.