Numerical simulation using the finite differential method was carried out to analyze the diffusion of an impulse sample in the micro-channel driven by electroosmosis.The results show that the electrical field strength applied externally and the concentration of buffer solution play a significant role in the diffusion of sample, however, hydraulic diameter and aspect ratio of height to width of channel play a small role in it. Weakening the electrical field strength applied externally and the concentration of buffer solution properly can prevent the sample band from broadening effectively, and promote the efficiency of testing and separation as well as keep a faster speed of transport. The conclusions are helpful to the optimal design for micro-channel.