Objective To establish a method for simultaneous determination of 50 kinds of antibiotics in dried sea cucumbers by multifunctional purification column combined with ultra performance liquid chromatography-tandem mass spectrometry. Methods The sample was extracted with 50% acetonitrile aqueous solution. After purification by multifunctional purification column, 50 kinds of antibiotics were separated using Waters ACQUITY UPLC HSS T3 (100 mm×2.1 mm, 1.8 μm) column with acetonitrile and 0.1% formic acid aqueous solution (include 5mmol/L ammonium acetate) for gradient elution. The mass spectrum was performed by electrospray positive ion mode scanning and multi-reaction monitoring acquisition, and quantified by external standard method. Results The results showed that 50 kinds of antibiotics exhibited good linearity in the range of 0.1-100.0 ng/mL, and the correlation coefficients were all greater than 0.99. The limit of detection ofthe method was 0.1-2.0 µg/kg, the limit of quantitation was 0.5-5.0 μg/kg. Recovery tests were carried out at 3 levels (1 times, 2 times, 10 times of the limit of quantitation) in the blank matrix, the average recoveries of the limit of detection were in the range of 54.83%-120.01%, and all the relative standard deviations (n=6) were 0.08%-9.15%. Conclusion This method has the technical advantages of simple operation process, excellent detection sensitivity, reliable precision, and good reproducibility, it can be applied to rapid screening and qualitative and quantitative detection of multiple antibiotics in dried sea cucumbers.