Pesticides are one of the most pollutants in aquatic ecosystems. Monitoring of aquatic pollution with these compounds is very important. Therefore, health status of aquatic animals can be used as a bio-indicator for pollution monitoring in surface waters. Diazinon is an organophosphorous pesticide, which is found in the aquatic environment of Iran. The concern has arisen over the ability of this compound to affect the health of fish in recent years. In this study, the effects of exposure to sub-lethal concentrations of diazinon on some biochemical parameters of rainbow trout, Oncorhynchus mykiss were examined after 7, 14 and 28 days. Acetylcholinesterase (ACEh) activity and the levels of total protein, albumin as well as globulin in plasma were significantly reduced at 0.1 and 0.2 mg/L concentrations tested (p<0.05). Creatine kinase (CK) activity was significantly lower in 0.1 mg/L diazinon group at 14th and 28th sampling periods, whereas its activity significantly increased in fishes exposed to 0.2 mg/L diazinon only on 7th day (p<0.05). Aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities and glucose levels in diazinon treated groups were significantly higher than the controlled group at experimental periods (p<0.05). Alkaline phosphatase (ALP) activity only significantly increased after 14 days in both diazinon treatments (p<0.05). The hypertrophy of hepatocytes, the vacuolization of cell cytoplasm, as well as hepatocyte cloudy swelling were observed in liver tissue of fish exposed to both concentrations of diazinon. Therefore, measurement of biochemical markers and liver tissue pathology studies are proposed as simple and suitable tools for assessing pesticide effects on fish.