Simultaneous determination of 13 kinds of sulfonamides and trimethoprim residues of aquatic by ASE-GPC-HPLC method. Pretreatment of fish samples using accelerated solvent Extraction. Extract concentrated by nitrogen blow with ethyl acetate: cyclohexane (1:1, V / V) mixture of 8mL constant volume.Automatic gel permeation chromatography Purification. Collect liquid nitrogen enrichment 1mL mobile phase constant volume, detemined by HPLC and quantified by external standard. The 13 kinds of Simultaneous and trimethoprim have good linear relationship in the range of 0.01 ~ 1.00mg/L and 0.02 ~ 2.00mg/L , the correlation coefficients are all above 0.9999. shrimp spiked with 100,200,400μg.kg-1 three concentration levels of 13 kinds of sulfonamides and trimethoprim mixed standard solution,the average recovery rate of standard are between 77.9%~98.6%, RSD is between0.4% ~ 13.4%. Developed a new method of 13 Sulfonamides and trimethoprim in aquatic products with ASE and GPC. the method was simple and accurate, 14 drugs were separate completely.