This study was performed to determine the effect of B1 and B12 vitamin injection on milk production, milk composition and some blood biochemical in Holstein dairy cows. A total 40 Holstein dairy cows with average milk production 30±50 kg, initial weight 600±50 kg and 28 days after calving were evaluated. The treatments were control diet, and intramuscular injection of 15 IU vitamin B1, 10 IU vitamin B12 or combination of vitamin B1 and B12 on a weekly basis. Milk production and its composition were measured after 4, 6, 8 and 14 week. Data from this study showed that although milk production was not influenced significantly The results of the study indicated that milk protein was increased significantly in B1 and B2 treated groups while milk lactose decreased significantly in B1. Total protein increased significantly in B1 while cholesterol and triglyceride increased significantly in B12 in lactating cows. The results indicated that treatment of vitamin B1 and B12 impacted milk composition and blood biochemical profile in lactating Holstein cows.