The objective of the present study was to investigate the effect of alfalfa seed oil (ASO) supplementation levels on growth performance, some blood metabolites and muscle fatty acids profile in broilers. A total of 600 one-day-old, unsexed Cobb broilers chicks were divided into four equal experimental groups (150 birds), each included three replicates of 50 chicks. The first treatment was control without any additives in feed; however, from second to forth group were supplemented with 250, 500, 100 g/ ton feed of ASO. Body weight, body weight gain and mortality rate were not influenced by alfalfa seed oil supplementation. On the other hand, feed intake was significantly (P=0.05) decreased and thus, feed conversion ratio (FCR) was improved significantly (P=0.03). Supplementation of ASO in broiler diet did not affected the carcass, heart, liver, gizzard and breast and thigh muscle relative weights, while abdominal fat relative weight was decreased significant (P=0.03) by added 1000 g ASO /ton feed. Plasma total protein, albumin, globulin, glucose and the activity of glutamic oxaloacetic transaminase (GOT) and glutamate pyruvate transaminase (GPT) were not affected by alfalfa seed oil supplementation levels, while plasma total cholesterol was significantly (p=0.002) decreased. Furthermore, the muscle contents of palmitic, oleic acid and liver MDA were not statistically (P> 0.05) affected by dietary alfalfa seed oil. However, the muscle contents of linolenic acid and vitamin E were significantly increased by increasing ASO supplementation level from 250 to 1000 g /ton feed, compared to the control group. Therefore, it could be concluded that alfalfa seed oil supplementation to broiler diets improved growth performance, modified plasma cholesterols and muscle fatty acids profile in broilers.