Abstract: A method for the simultaneous determination of residues of 5 macrolide antibiotics in surface water around pharmaceutical enterprises, including tylosin, josamycin, spiramycin, noramycin and roxithromycin, was proposed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with automatic solid phase extraction. The surface water samples around the pharmaceutical enterprises were collected randomly and stored in cold storage. 500 mL of the surface water sample was taken, and adjusted the acidity to neutral. The above water sample was passed through the activated HLB solid phase extraction column at a flow rate of 15 mL·min^-1, and eluted with 16 mL of acetone. The eluent was collected and blown to nearly dryness by nitrogen, and the residue was dissolved and made its volume up to 1 mL with methanol. The 5 macrolide antibiotics in the obtained solution were separated on Shimadzu Venusil MP C₁₈ column, using a mixture of methanol-0.1% (volume fraction) formic acid solution at different volume ratios as mobile phase for gradient elution. Multi-reaction monitoring mode was adopted in MS analysis. As shown by the results, linear relationships between values of the corresponding peak area and mass concentration of 5 macrolide antibiotics were found in the range of 0.001-5.0 mg·L^-1, with detection limits (3S/N) in the range of 0.14-0.25 μg·L^-1. Test for recovery was made by the standard addition method, giving results in the range of 85.4%-96.6%. The 6 spiked water samples (0.01 mg·L^-1) were prepared, with RSDs (n=6) of determined values less than 5.0%.