Actually the kinetic study was carried out to standardize the method of estimation and to fix the parameters such as temperature, pH, substrate concentration, and time and enzyme concentration for studying entire reproductive cycle pertaining to β-glucuronidase activity. In Labeo rohita, the reproductive cycle was classified on the basis of gonadosomatic index and seasonal variation in the testes (morphological and histological variation). It was observed that the β-glucuronidase activity increased in the kidney and liver from preparatory to spawning phase and was lowest in the postspawning. In testes, the lowest activity was recorded in the postspawning and the highest in the prespawning and early period of spawning phase. It has been concluded that the sex regulating hormones may be playing role in the modulation of this enzyme activity in the studied organs during its reproductive cycle. Further, this enzyme may not have any role in the degeneration of testes during post-spawning phase and therefore the activity in the testes decreases during post-spawning as compared to spawning phase.