Background: γ-glutamyltransferase(EC: 2.3.2.2) activity increased in hepatocellular diseases in animals and human including sarcocystosis. Objectives: The present study aimed to investigate the activities of hepatic enzyme; alanine transaminase (ALT), aspartate transaminase (AST), and gamma glutamyltranspeptidase (GGT) during the sarcocystosis infestation in buffalo. Also, to explore the purification, kinetic properties, and effect of anticoccidial drug ambrolium on the most affected enzyme. Methods: Blood and liver samples were collected from 10 sarcocyst-free and 15 sarcocyst-infested buffalos from Alexandria abattoirs, Egypt. Results: The result revealed that the specific activities of plasma ALT and AST were non-significantly increasedbut, GGT significantly increased in infested buffalo. Hepatic ALT, and GGT specific activities were significantly decreased while, AST activity was increased in infested-buffalo. The purified GGT enzyme had lowered specific activity in infested buffalo than control one and the enzyme consist of two subunits with molecular weight of 29.000 and 70.000 dl. Also, the purified enzyme from infested-buffalo had higher Km and lowered Vmax as compared to control one. The optimum temperature of purified GGT was 37 ºC and the ambrolium drug had an inhibitory effect on purified enzyme. Conclusion: We concluded that, the alteration in hepatic enzymatic activities considered a biomarker for diagnosis of sarcocystosis in buffalo, and ambrolium had an inhibitory effect of competitive type on purified enzyme