Accurate estimation of the kinetic parameters is essential for on-line optimization and control in bio-catalytic reactor system. Fructose production from glucose using immobilized enzyme was chosen as a model system and deactivation constant which is the most sensitive among the kinetic parameters was estimated from the measure-ments of product concentration. The effects of sensitivity on the sequential parameter estimation were analyzed under the operating conditions of both constant temperature and varying temperature. By considering the parameter sensi-tivity on Kalman filtering, estimated values showed reduced fluctuations in the initial period of operations where the sensitivity was low and better convergence to the true value as the sensitivity increased.