Abstract: The alkaline phosphatase from Oreochromis niloticus (Tilapia) were selectively modified by chemical reagents such as PMSF, PCMB, NBS, TNBS, SUAN, DTT, IAc and BrAc respectively. It could be found that the enzyme activity was significantly decreased after modification of PMSF, NBS, but oppositely the PCMB, TNBS, SUAN, DTT, IAc, BrAc didn爷t obviously effect the alkaline phosphatase enzyme activity. Studied of the chemical modification demonstrated that the Tryptophan residues and Serine residues were essential functional groups inside of the alkaline phosphatase active site, the same time suggested that the others residues were not located in the enzyme active site, didn’t directly effect the enzyme activity.