The effect of ascorbic acid on shelf life of rainbow trout (Oncorhynchus mykiss) fillets in the refrigerator based on total microbial load, chemical evaluation was investigated. For this reason, the fish were divided into three groups, including without injections (treatment 1), fish with intravenous injection of ascorbic acid in doses of 0.1 and 0.3% (treatment 2 and 3) and fish with intramuscular injection in doses of 0.1 and 0.3% (treatments 4 and 5). After 62 hours of injection, fish (400 ±10g) were caught and filleted, then kept for 16 days in refrigerator. During this period, every 4 days, peroxide value (PV), thiobarbituric acid (TBA), free fatty acids (FFA), total volatile nitrogen base (TVB-N) and total bacterial load (TVC) indicators were measured. Treatments 3 and 5 showed a significant difference (p≥0.05) in PV, TBA, FFA, TVB-N and TVC indicators in comparison to other treatments. There was also a significant difference between the treatments 3 and 5 (p≥0.05) in the indicators of the TVC, TBA, FFA and TVB-N that indicated better performance of intravenous injection of ascorbic acid 0.3% after 8 days storage in comparison to intramuscular injection of 0.3%. According to the results, in intramuscular or intravenous injection of different doses of ascorbic acid increased the shelf life of rainbow trout fillet during storage in the refrigerator until day 12.