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EcoRI (pronounced, "eco R one") is a restriction endonuclease enzyme isolated from species E. coli. The Eco part of the enzyme's name originates from the species from which it was isolated, while the R represents the particular strain, in this case RY13. The last part of its name, the I, denotes that it was the first enzyme isolated from this strain. EcoRI is a restriction enzyme that cleaves DNA double helixes into fragments at specific sites. It is also a part of the restriction modification system.

In molecular biology it is used as a restriction enzyme. EcoRI creates 5 nucleotide sticky ends with 5' end overhangs of AATTC. The nucleic acid recognition sequence where the enzyme cuts is G/AATTC, which has a palindromic, complementary sequence of CTTAA/G. The / in the sequence indicates which phosphodiester bond the enzyme will break in the DNA molecule. Other restriction enzymes, depending on their cut sites, can also leave 3' overhangs or blunt ends with no overhangs.