In this study, used cooking oil, ethanol and sodium hydroxide (NaOH) were used as raw material and catalyst to produce biodiesel through transesterification reaction. The occurring emulsion from ethanol acts as a surfactant in the reaction mixture make the separation of biodiesel from glycerol layer more difficult. This problem solving was done by adding pure glycerol into the reaction mixture after complete reaction to make quick layer separation between glycerol and biodiesel. The characterization of the produced biodiesel was performed by proton nuclear magnetic resonance ( 1 H-NMR) and gas chromatography (GC). The result showed that the optimum condition for ethyl ester biodiesel production were 8:1 of ethanol to oil molar ratio, 0.75 %w/w of NaOH amount, 45 min of reaction time and 40 o C of reaction temperature. This condition gave 100% of fatty acid ethyl ester conversion that determined by 1 H-NMR. The result on the optimum condition for layer separation showed that the adding of 2.44% v/v of pure glycerol relation to the total volume of the reaction mixture took 14.38 min for glycerol-biodiesel layer separation. In addition, ethyl ester biodiesel was further analyzed its fuel properties followed ASTM biodiesel standard. The results showed the produced biodiesel met well with the standard.