At a water temperature of (23±2) ℃, Spinibarbus caldwelli was treated with a single oral administration of neomycin sulfate at a dosage of 60 mg/kg body weight. The concentration of neomycin sulfate in the muscle, liver, and kidney of Spinibarbus caldwelli was determined using HPLC-MS/MS, and the residues and degradation patterns of neomycin sulfate in Spinibarbus caldwelli were studied. The results revealed that after a single dose of oral administration of neomycin sulfate, the elimination half-life T1/2ke of neomycin sulfate in the muscle, liver, and kidney of Spinibarbus caldwelli were 44.62 h, 248.50 h, and 616.90 h, respectively. This indicated that neomycin sulfate was eliminated most rapidly in the muscle, followed by the liver, and slowest in the kidney. Muscle was used as the target tissue for residual detection. According to the current National Food Safety Standard-Maximum Residue Limit of Veterinary Drugs in Food (GB 31650—2019), the maximum residue limit of neomycin sulfate in all food animal muscles is set at 500 μg/kg. Therefore, it is recommended to refrain from consuming Spinibarbus caldwelli until at least 10 d have passed under the experimental conditions to ensure safety. ［Chinese Fishery Quality and Standards, 2024, 14(2): 01-06］