The method was established for determining the related substance and its chirality enantiomer of emtricitabine.The method was performed on related substance with SHIMADZU VP-ODS chromatog. column (150 mm × 4.6 mm, 5 μm); flow liquid was 0.02 mol/L-1 ammonium acetate buffer solution (ammonium acetate 1.54 g, adding water to 1000 mL, using acetate to regulate pH value to 3.85-3.95)-methanol (85:15); flow rate was 1 mL/min-1, and determination wavelength was 280 nm.The method was performed on chirality drug with CHIROBIOTIC TAG chirality chromatog. column (macrocyclic glycopeptide, 5 μm, 250 mm × 4.6 mm); flow liquid was methanol-triethylamine-acetic acid (100:3.5:2.4); flow rate was 1 mL/min-1, and determination wavelength was 280 nm.The detection limits was 0.1443 ng for related substance.The degradation peaks produced by destroy emtricitabine with strong acid, alkali, high temperature, and oxidation resp. could be well separated from that of emtricitabine by this method.The limits was 0.45 ng and the resolution was 1.6 for chirality enantiomer.This is a rapid accurate, reliable, and sensitive method for detecting related substance and chirality enantiomer of emtricitabine.