To solve the interference problem of undesired metabolites with complex background fermentation liquid， high performance liquid chromatography method for analyzing suitable for in vitro fermentation conditions， the measurement daidzein and equol. The fecal microbiota was transformed into daidzein in Brain Heart infusion (BHI) broth medium for 48 hours. After extracted by ethyl acetate， the samples were analyzed on a C18 column (250 mm × 4.6 μm， 5 μm). The flow rate was 0.8 mL/min， the column temperature was 25 ℃， the injection volume was 20 μL， and the mobile phase was water-methanol -acetonitrile (volume ratio， 5∶3∶2). The detection was carried out at UV 230 nm and 260 nm. The linear correlation coefficients of daidzein and equol were R2 = 0.9999， 0.9996， respectively， the average recovery were 92.12%-104.34%， the relative standard deviation were 0.15%-0.24%， the limit of detection were 0.07 μg/mL and 0.05 μg/mL， and the limits of quantification were 0.23 μg/mL and 0.16 μg/mL， respectively. The method established in this study had high accuracy and precision and was simple. It has been applied to determine daidzein and equol in fecal microbiota fermentation.