A high performance liquid chromatography (HPLC) method for the determination of trace methanol in high water content sample has been established. Using 4-[N-methyl, N-(1-naphthylmethyl)]-amino-4 oxobutanoic acid (NAOB), 4-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) under a mild condition, the derivatization of methanol in aqueous solution was smoothly carried out before HPLC analysis. After derivatization, acidic or basic interfering substances and excess reagents can be easily removed from organic layer by simple acid-base shake-out. The condition for optimal derivatization of methanol in aqueous solution was established and applicable to the analysis of commercial Chinese liquor medicine (ethanol content 8~55%, v/v), the range of trace methanol in the above samples was 2.20 × 10-4~ 3.28 × 10-3% (v/v). The result showed that methanol contents in all samples were below regulation standard (< 0.1%, v/v).