Abstract: 　　To establish an accurate and efficient liquid chromatography-tandem mass spectrometry (LC-MSMS) method for the determination of levomethorphan,dextromethorphan and its metabolite dextrorphan in hair and urine. Hair samples were extracted by grinding and ultrasonic with the methanol solution containing the internal standard dibenzoyl-ethylenediamine (SKF_525A), urine samples were extracted by protein precipitation with internal standard diazepam-D5. Column was Astec CHIROBIOTIC^@ V2 (2.1×250 mm, 5 µm) chromatographic and the column temperature was 25°C, mobile phases were 0.5‰ formic acid (10 mmol/L ammonium acetate) and methanol, gradient elution at 0.4 mL/min. Detection was performed in positive ion multiple reaction monitorin (MRM) mode. The limits of detection (LOD) for levomethorphan, dextromethorphan, and dextrorphan were 0.05 ng/mg in hair and 0.5 ng/mL in urine, the limits of quantification (LOQ) were 0.1 ng/mg in hair and 1 ng/mL in urine. Good linearity was observed in the concentration ranges of 0.1 ng/mg～10 ng/mg for hair and 1 ng/mL～200 ng/mL for urine with correlation coefficients over 0.99. Besides, accuracy, precision, matrix effect, recovery and dilution reliability all met the methodological requirements. High concerations of dextromethorphan and dextrorphan were detected in hair and urine samples from actual cases. This method was simple, rapid, highly sensitive and reliable for the detecting of levomethorphan, dextromethorphan and its metabolite dextrorphan in hair and urine.