Objective To filter the noises in the experimental data of parallel plate flow chamber for observing more clearly the events occurring in the process of cell rolling adhesion and develop a new method to measure the elasticity of microvillus on cells based on the flow chamber experiment. Method The experiment of E-selectin regulated HL-60 cell rolling was performed by flow chamber system, and the data were denoised by wavelet analysis so that the high frequency thermal response signals were extracted from the data. Based on the equipartition theorem and equilibrium equations of tethered cell, the relationship between the cell microvillus spring constant and thermal fluctuations was constructed. Results Filtering noises from cell rolling time course by wavelet analysis, the events such as free rolling, slowing down, stopping and speeding up of rolling cell could be observed more easily; almost 80% of fluctuating energy of a rolling cell was involved in its high frequency fluctuation which was regarded as the thermal response of the cell to the Brown movement of water molecules, and the spring constant of microvillus on HL-60 cell was measured to be (13.7±7.4) μN/m at wall shear stress from 0.01～0.06 Pa. Conclusions The wavelet analysis can filter the thermal noises in cell rolling data of flow chamber experiment, and since the rigidity information of cell microvillus is involved in and can be extracted from the high frequency thermal fluctuation of the rolling cell, the parallel plate flow chamber experimental technique can be extended to measure the elasticity of microvillus on cells.