This study was conducted to evaluate seminal enzyme (GOT, GPT and LDH) profile and its relationship with quality of buffalo frozen spermatozoa. For this purpose, twenty-five split pooled ejaculates from five buffalo bulbs in Buffalo Breeding and Training center (Jabal-Urmia) possessing more than 80% visual sperm motility, were diluted at 37oC in tris- egg yolk extender. The diluted semen was cooled to 4oC within 2 hours, equilibrated at 4oC for 4-6 hours following the addition of glycerol, filled in 0.5 ml French straws and were subjected to cooling condition before being plunged into liquid nitrogen. Semen was thawed at 37oC for 40 seconds after 72 hours of storage inside liquid nitrogen. Post thaw visual sperm motility, plasma membrane integrity and acrosome morphology of each frozen semen sample were assessed by warm plate microscopy at 37oC, hypo osmotic swelling test, and Giemsa staining, respectively. LDH and GOT (AST), GPT (ALT) enzyme activities were measured using kinetic oxidation of lactate to private method and colorimetric method, respectively. Overall mean activity of frozen semen GOT (AST), GPT (ALT) and LDH enzyme activities were 140.4±18.5 (IU/L), 270.4±58.5 (IU/L) and 90.4±32.7 (IU/L), respectively. The relationship between the activities of these enzymes and semen quality showed only a positive correlation between increased LDH and GOT (AST), PT (ALT) enzyme activities and decrease post thaw spermatozoa characteristics. These result revealed that seminal enzyme (GOT, GPT and LDH) profile in extender is suitable and recommendable for evaluation of buffalo frozen semen.