A sensitive and robust HPLC method was developed for the determination of kanamycin (Kan) in swine tissues. Kan was extracted from tissue with 10% TCA (w/v) solution and cleaned up with MCX SPE column. The purified extract was derivatized with 9-fluorenylmethyl chloroformate, and then separated on a C18 column and detected by a fluorescence detector. The linear range of the calibration curve was 0.025-1 microg/mL with R(2) of 0.9998. The limits of quantification were determined to be 0.1 mg/kg in muscle, 0.2 mg/kg in liver, and 0.6 mg/kg in kidney, and the average recoveries of Kan from swine tissues at different fortification levels (0.1-0.4 mg/kg for muscle, 0.2-1.2 mg/kg for liver, and 0.6-5.0 mg/kg for kidney) ranged from 80.7 to 91.3% with intraday and interday coefficient of variations less than 12.1%. The validated method was successfully applied to determine Kan in incurred samples, indicating that it can be used as a routine tool for the surveillance of Kan residue in swine tissues.