A sensitive and selective method was developed for the determination of microcystin-LR (MC-LR) based on proteasomal trypsin-like activity inhibition. Under the optimized conditions, the detection limit for MC-LR was 0.18μg/L, The linearity range was 0.2-2 μg/L，and the average recovery for MC-LR in tap water was 128%. Microcystin-YR (MC-YR) and Microcystin-RR (MC-RR) have no inhibiting effect on the proteasomal trypsin-like activity in the concentration ranged between 0.1-100 nmol/L. This method could be applied for the selective, high-throughput, and rapid determination of MC-LR in waters.