Aim: To establish a sensitive HPLC method for the determination of aldose reductase (AR) activity, based on the HPLC analysis of NADP derivative fluorescence formed by AR. Methods: AR was isolated and purified from rat kidney. The mobile phase was acetonitrile-water (75:25) delivered at a flow rate of 1.0 ml·min by isocratic reversed-phase elution on C column. The temperature was controlled at 25°C and the fluorescence of NADP derivative was monitored at 370 nm/465 nm. The effect of NADPH, DL-glyceraldehyde, pH, and temperature on AR activities was studied. Results: The NADP derivative was stable, whose retention time was (1.4 ± 0.01) min with no interference peaks. A good linearity was observed (r = 0.9994) with the detection limit at 25 pmol. Both the inter-day and intra-day precision was less than 10%. Conclusion: The method is simple, sensitive and credible and can be used for the determination of AR activities.