To develop a spectrophotometric method for determining the concentration of recombinant hirudin (rH) in urine of rats. rH concentration was determined based on the rH inhibility to thrombin which hydrolyzed the Chromozym TH TH chromogenic substrate to form the specific pNA absorbed at 405nm. The standard rH in rat urine was determined by the spectrophotometric method at concentration of 6.25 to 75 ng·mL-1 with day and intra-day RSD <10%, method recoveries of >95% and the dilution recoveries of >93%. The rH samples of rat urines which iv dose of 0.5, 1.0, and 2.0 mg·kg-1 were collected and analyzed by the CSA method. Their cumulative excretion rH at 0～12 hr were (116.850±57.160), (235.544±39.375) and (474.986±85.426) μg·kg-1. The calculated cumulative excretion rate of three doses is about 23% which indicates that the rH was eliminated in the way of a linear kinetics in rats. The rH content in rat urine could be measured by the spectrophotometric method accurately, reliably and sensitively for the rH urinary excretion dynamics study.