In the present study the production of biodiesel was performed by using raw material like soybean oil by trans-esterification process. According to the American Society for Testing and Materials (ASTM), the international specification free glycerol in biodiesel should not be more than 0.02 mass %. To achieve the biodiesel with the ASTM specification, biodiesel was separated using prepared PAN ultrafiltration membranes. The polyacronitrile ultra filtration membranes were prepared on supporting material of woven fabric by phase inversion technique of membrane casting. The prepared membranes were characterized in terms of its molecular weight cut off and flux of the membranes. Different molecular weights of the BSA solutions were used to determine the molecular weight cut off of the membranes. Then the obtained 6KDa and 15KDa Ultra filtration PAN membranes were used to separate the glycerol from (FAME) free acid methyl ester. It was observed that the both membranes were separated glycerol from the biodiesel below 0.02 mass % which meets the requirements of the ASTM specification of glycerol. The permeate side of the 6KDa membrane was estimated to be 0.017 mass % of glycerol, whereas, 15 KDa membrane was 0.02 mass % . The glycerol percentage in retained side membranes were increased with time.