This paper describes kinetic parameters of an enzymatic reaction obtained by measuring the reaction rates of hydrolysis by trypsin at various flow rates and temperatures. Using these parameters, the mechanisms of an efficient reaction of nonimmobilized, i.e., "free state" enzyme in microchannel laminar flow can be examined from a kinetic perspective. Efficient complex formation between the enzyme and substrate was confirmed through Michaelis-Menten analysis. Results show that the enhancing effect of microfluidics in complex formation accelerated the enzymatic reaction. Results of activation energy analysis confirmed that a decrease in the apparent activation energy occurred as the flow rate increased. Taken together, these results suggest that a microfluidic system is useful for performing rapid reactions by influencing the complex formation and activation energy.