The concentration of diethylstilbestrol (DES) in the microbial degradation systems was determined using HPLC—UV. Different extraction methods were used in the fungi and bacteria fermentation systems, DES was separated through a Sepax GP-C₁₈ (150 mm×4.60 mm, 5.0 μm), eluted with methanol-water (7︰3, V/V) at 0.7 mL/min, and detected at 242 nm. Results showed that retention time of DES was 7.714 min. Good linearity was obtained with the detection limits of 0.5～50.0 mg/L with a correlation coeffcient of 0.996 3. The average spike recovers of DES for bacteria and fungi fermentation systems were 94.04% and 95.24%, respectively, with RSD of 2.33% and 1.05%, respectively. The limits of detection was not more than 0.30 mg/L. Results showed that the ratios of DES (25 mg/L) degration of four strains could degrade 62.38%, 64.72%, 53.38%, 88.34% , respectively in 5 d. The developed method presented the benefits of simplicity, rapidity, good separation, high accuracy, high precision and good stability.