EcoRI (pronounced, "eco R one") is an endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system.

In molecular biology it is used as a restriction enzyme. It creates 4 nucleotide sticky ends with 5' end overhangs of AATT. The nucleic acid sequence where the enzyme cuts is GAATTC, which, as the complementary sequence is CTTAAG, has rotational symmetry.