Abstract: Objective: In this study, a one-step purification method combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) was established for the simultaneous detection of 54 veterinary drug residues belongs to 13 classes of ya-fish (Schizothorax prenati). Methods: 8 mL of 0.2% formic acid-acetonitrile:water solution (90:10, V/V) was added to 2.0 g sample and extracted by ultrasonication in an ice-water bath and cleaned up in one step by LPAS. The determination was performed on a C₁₈ column with a gradient elution of methanol-0.1% formic acid-5 mmol/L ammonium acetate solution, dynamic multiple reaction monitoring (dMRM), positive/negative ionization mode, and quantitative LC-MS/MS by internal standard method. By adding 54 veterinary drugs, the extraction method, extraction volume, salt addition, liquid phase and mass spectrometry conditions were optimized, and the method performance (matrix effect, detection limit, quantification limit, accuracy and precision) were verified. Results: The linear relationships between the 54 drugs and their concentrations were good within the range of 0.1~50 μg/L, coefficient of determination (R²) ≥0.992. The limits of detection (LODs) were between 0.03~2.5 μg/kg and the limits of quantification (LOQs) were 0.1~8.3 μg/kg. The average recoveries of the 54 drugs at low (5~10 μg/kg), medium (10~20 μg/kg) and the high level (50 μg/kg) ranged from 70.45% to 118.1%, with relative standard deviations (RSD) between 0.01% and 9.9%. This method was applied to detect the drug in 50 samples of cultured ya-fish, and drugs were detected in seven of them. Enrofloxacin and danofloxacin were each detected twice, and sulfamethoxazole was detected four times, all at levels below the limit of quantification. Conclusion: The method was simple to operate, one-step purification, and had low limits, high sensitivity, good accuracy and precision, which met the technical requirements of multi-veterinary drug residue detection, and can provide technical support for the rapid and efficient detection of various veterinary drug residues in large quantities of ya-fish.