Ozone has a strong oxidation power that allows effective inactivation of waterborne viruses. Few studies have accurately measured the kinetic relationship between virus inactivation and ozone exposure, because the high reactivity of ozone makes it difficult to measure them simultaneously. A continuous quench flow system (CQFS) is a possible solution for analyzing such a fast reaction; however, previous studies reported that CQFS provided different results of inactivation rate constants from the batch system. The objectives of this study were (1) to develop a CQFS to evaluate the kinetics of microbial inactivation accurately, (2) to evaluate the inactivation rate constants of waterborne virus by ozone, and (3) to compare the results with previous studies. The results indicated that the simple plug flow assumption in the reaction tube of CQFS led to underestimation of the rate constants. The accurate measurement of rate constants was achieved by the pseudo-first-order reaction model that takes the residence time distribution (RTD; i.e., the laminar flow assumption) into account. The results of inactivation experiments suggested that the resistance of viruses were getting higher in the following order: Qβ < MS2, fr, GA < CVB5 Faulkner, φX-174, PV1 Sabin, CVB3 Nancy. The environmental isolates of CVB3 and CVB5 had a 2-fold higher resistance compared with their lab strains. Predicted CT values for 4-log inactivation ranged from 0.018 mg sec L−1 (Qβ) to 0.31 mg sec L−1 (CVB3 Environmental strain). The required CT values for 4-log PV1 inactivation was 0.15 mg sec L−1, which was 166-fold smaller than those reported in the United States Environmental Protection Agency guidance manuals. The overestimation in previous studies was due to the sparse assumption of RTD in the reactor. Consequently, the required ozone CT values for virus inactivation should be reconsidered to minimize the health risks and environmental costs in water treatment.