The relationship between the dynamic effect of a capillary and the length of microchannel in front of a step valve was researched, then the influence of dynamic effect in the process of stopping the capillary in microchannel formed by hydrophobic and hydrophilic walls on the step valve was analyzed. According to the working principle of the passive valve of capillary and energy conservation law, the critical length in front of the step valve was calculated when the step valve stoped the capillary. The calculated length corresponding to the critical length was obtained by numerical simulation. It shows that when the actual length in front of the step valve is greater than or equal to the calculated length, the valve can stop the capillary effectively. The step valves were fabricated by bonding polydimethylsiloxane (PDMS) to glass. Experiments of stopping the capillary by the step valve were performed in a rectangular microchannel formed by three hydrophobic PDMS walls and one hydrophilic glass wall. For the series of microchannel with a depth of 40 μm and widths from 200 μm to 400 μm, the calculated length in front of the step valve is from 4.531 6 μm to 10.081 μm. In the experiments, the actual length before the step valves for effectively stopping the capillary is in the interval from 10 μm to 2 000 μm. The results show that even if the actual length in front of the step valve is 10 μm, the step valve can also stop the capillary. Therefore, the dynamic effect of the capillary can be ignored in stopping the step valves in the microchannel.