Objective To evaluate on-line clearance monitoring (OCM) of urea in the assessment of Kt/V during hemodialysis. Methods Kt/V values of 23 maintenance hemodialysis patients treated in this hospital were assessed by OCM and by single-pool urea kinetic model. The Kt/V values by the two methods were compared. The Kt/V values form the patients using the dialyzer for the first time and at the 7th time were also compared. Results In patients who used the dialyser for the first time, the Kt/V values were (1.25±0.16) by OCM and (1.37±0.23) by single-pool urea kinetic model (r=0.842, P <0. 001). In those who reused the dialyser at the 7th time, the Kt/V values were (1.23±0.15) by OCM and (1.31±0.17) by single-pool urea kinetic model (r=0.667, P＜0.01). The Kt/V values from patients using the dialyzer for the first time and those from patients reusing it at the 7th time had no significant differences both by OCM (t=1.908，P＞0.05) and by single-pool urea kinetic model (t=1.692，P＞0.05). Conclusion Kt/V values by OCM are usually lower than those calculated from urea measurements. This difference has to be considered in application of the results to clinical practice. The dialyser reused for 7 cycles still has unchanged clearance ability to small molecule solutes.