An optimized method based high-performance liquid chromatographic (HPLC) was developed for the determination of free gossypol content in cottonseed meal. The gossypol in cottonseed meal was extracted with acetone by ultrasound, centrifugation, suction filter, rotating evaporation, and then dissolved with the mixture of acetonitrile and 0.2% phosphoric acid (85: 15, V/V). The optimal conditions of HPLC were as follows: the column was Agilent TC-C₁₈ (250 mm×4.6 mm, 5 μm), and mobile phase was the mixture of acetonitrile and 0.2% phosphoric acid (85: 15, V/V) with a flow rate of 1.0 mL/min. UV detected wavelength was set at 235 nm, the injected sample volume was 20 μL and the column temperature was 25 ℃. The results showed that the equation of the standard curve was y=189 347x+24 969 (r=0.999 99), which had a highly linear relationship when gossypol content ranged from 0.122 4 μg/mL to 78.336 μg/mL, and the minimum detection limit of gossypol was 0.52 mg/kg. The relative standard deviation of gossypol extracted from cottonseed meal stability at 4 ℃ within 36 h was 1.51% to 3.43%. The average recovery rate of the method was 94.72% to 99.49% with relative standard deviation of 1.72% to 3.13%. In conclusion, the results show that the proposed HPLC method is of good stability, accuracy, sensitivity, and high efficiency.