The Phase Shift Micro XAM-3D was used to measure the roughness of smooth wall, new lateral wall and lateral wall attached by biofilm. The phaseDoppler particle anemometer was used to measure the particle movement with the effect of different walls. The study used three different particle sizes: 10μm, 50μm, 100μm under three different walls. The results showed that the surface area of biological lateral wall (212%) was significantly higher than that of new lateral wall (4.37%) and smooth wall (0.196%). The velocity of small particles (particle size of 10μm) collided with smooth wall was lower than that collided with the biofilm lateral wall. The velocity distribution of smooth wall, new lateral wall and biofilm lateral wall tended to be gradually wider and wider. While the average velocities of middle particles (50μm) and big particles (100μm) are closed to zero and didn’t widen. The reason is that the biofilm enhanced the surface adsorption capacity which reduced the number of small particles. The result proved that the effect of biofilm on big particles was not significant.