A method of determination of ciprof loxacinum in the hemolymph of Chinese mitten-handed crab, Eirocheir sinensis, was established by RP-HPLC. The drug was administered by intramuscular at a dose of 8. 17 ? 0. 56 and 6. 25 ? 0. 85 mg#kg - 1 to male and female crab, repectively . The hemolymph was extracted from the cardiac region. By comparison among various ant-i coagulant methods, the permanent result of ant icoagulation was obtained through ACD mixing with equal volume hemolymph. Hemolymph samples got rid of protein by methanol, centrifuged at 3000 r#min- 1 for 10minutes and then the supernatant was used to HPLC. The RP-ODS- 5 LL 50 @ 4. 6mm ID column was used, with a mobile phase of methano-l phosphate buffer-tertrabutylammonium bromide= 250B750B20 ( v/ v/ v ) ; The f low rate was 1 mL#min - 1 ; the auto-injected volume, 2 LL and the uv detection wavelength, 280nm. The study showed that the linearity and repeatability were good; the average recovery of both male and female crab were : 100. 08 ? 3. 43% and 99. 11 ? 8. 20% ; the intra-day precision, the average extracted-precision were 1. 19 ? 0. 92%, 1. 55 ? 0. 01% and 2. 31 ? 1. 03% , respectively. The law of the metabolism of blood-drug concentration was disclosed dropping from 37. 12 ? 0. 08Lg#mL- 1 to 5. 96 ? 0. 18 Lg#mL- 1, by using RP-HPLC method to determine the ciprofloxacinum in hemolymph within 48 hours in male crab after administration of the drug through intramuscular.