A method for the separation and determination of Sarafloxacin by HPLC was developed. The sample was dissolved in a mixture of V(acetonitrile):V(water) = 1:1. Operating conditions were as follows: mu-Bondapak C18 column (3.9 mm x 300 mm), V(acetonitrile):V(methanol):V(2 mmol/L H3PO4, adjust pH 3.5 with triethylamine) = 30:5:65 as mobile phase with a flow rate of 1 mL/min, UV detection at 278 nm, and column temperature was 15 degrees C. Under the above conditions, Sarafloxacin and other impurities were separated from each other. The method was simple, rapid, sensitive and accurate.