This study was conducted to investigate the effects of alfalfa (Medicago sativa) extract on hepatic enzymes performance and serum biochemical parameters of rainbow trout (Oncorhynchus mykiss). In this regard, 750 fish (initial weight: 10. 4 ±,0. 5 g) distributed in concrete pools and fed by 5 dietary treatments including 0 (control), 0. 5, 1, 2, and 3 percent of alfalfa extract, respectively for 60 days. Serum parameters analysis showed that there was a significant difference between fish fed with alfalfa extract and control group. The highest and lowest levels of serum glucose were observed in treatment 4, and treatment 1 (control), respectively (p < 0. 05). The highest level of total serum protein was observed in treatment 2, and the lowest was observed in treatment 5 (p < 0. 05). However, there was no significant difference between control, treatments 2, and treatment 4 in terms of total serum protein level (p > 0. 05). The highest level of serum albumin was recorded in control. Based on the results, different levels of alfalfa extract led to a downward trend in serum triglyceride and cholesterol levels. The highest level of serum phosphorus was observed in treatment 4, and the lowest level was observed in the control (p < 0. 05). Serum magnesium levels were also affected by alfalfa extract (p < 0. 05). Based on the results, no significant differences were observed between experimental treatments in serum SGOT and SGPT levels during the 60-day experimental feeding period (p > 0. 05). The highest level of serum alkaline phosphatase (ALP) was in treatment 5 and the lowest level of serum ALP was observed in control (p < 0. 05). The highest level of serum lactate dehydrogenase (LDH) was observed in control and the lowest in treatments 3 and 4 (p < 0. 05). Based on the results, 2 percent of alfalfa extract in the diet had the best effects on serum biochemical parameters and hepatic enzymes.