Enzymatic hydrolysis of butter oil was carried out using Palatase 20,000 L as biocatalyst. The fatty acid components of butter oil and the hydrolysates of butteroil were analyzed using a combination of thin layer chromatography (TLC) and gas chromatography (GC). The flavor compounds were analyzed by SPME-GC-MS. Palatase 20,000 L -catalysis significantly changed the composition of fatty acids of the butter oil and contributed to releasing the flavor compounds, especially the key flavor compounds octanoic and decanoic acid (P<0.05). Therefore, Palatase 20,000L lipase-catalysis is a potential method for improving the nutritional value and flavor of butter oil.