Abstract: Waste water sample (500 mL) was adjusted to pH 2.0-4.0 with 6 mol·L^-1 hydrochloric acid solution, and 150 mg of ascorbic acid and 250 mg of ethylenediamine tetraacetic acid disodium were added. HLB solid phase extraction column was used for enrichment and purification, and 10 mL of methanol was used for elution twice. The eluate was dried by nitrogen blowing and internal standard solution (25 μL) was added. The solution was made its volume up to 1.0 mL with initial proportional mobile phase and filtered by 0.22 μm microporous membrane for test. ZARBAX Eclipse Plus C₁₈ chromatographic column was used as the stationary phase, and a mixture of formic acid, methanol, acetonitrile and water at different volume ratios was used as the mobile phase for gradient elution. ESI⁺ and MRM mode were used in MS analysis, and internal standard method was used for quantitative analysis. As shown by the results, the linear ranges of standard curves for 12 antibiotics were all 2.00-200 μg·L^-1, with detection limits (3.143s) in the range of 0.001-0.007 μg·L^-1. Recovery test was made on the blank sample at 3 concentration levels by standard addition method, giving results in the range of 60.1%-125%, and RSDs (n=6) of the determined values were in the range of 2.8%-13%. This method has been applied to the analysis of actual water samples, and the results showed that antibiotics were detected in 5 water samples from different sources in varying degrees.