Choline is important an organic compound for normal membrane function, acetylcholine synthesis, lipid transport, and methyl metabolism. In biological tissues and foods, there are multiple choline compounds that contribute to choline content. There are so many analytical methods for choline determination, such as radioisotopic, high-performance liquid chromatography, and gas chromatography/mass spectrometry. However, these existing methods are expensive, unmanageable, and time-consuming. In this study, we modified enzymatic method, which is applicable for the determination of choline in milk and infant formulas, and applied to bovine serum and muscle. The calibration curves were linear with higher correlation coefficients than 0.994 . Recoveries obtained by calibration curves from the spiked bovine serum and muscle samples varied between 70.6 and 85.2%. The method may be suitable for use as a routine method in the determination of choline for biological tissue and food samples.