In this paper, the effect of membrane features on flow characteristics in the microfluidic passive valve (MPV) and the membrane behavior against fluid flow are studied using the fluid-structure interaction (FSI) analysis. Firstly, the microvalve model with different numbers of microholes and pitches of microholes are designed to investigate the flow rate of the MPV. The result shows that the number of microholes on the membrane has a significant impact on the flow rate of the MPV, while the pitch of microholes has little effect on it. The constant flow rate maintained by the microvalve (the number of microholes n = 4) is 5.75 mL/min, and the threshold pressure to achieve the flow rate is 4 kPa. Secondly, the behavior of the membrane against the fluid flow is analyzed. The result shows that as the inlet pressure increases, the flow resistance of the MPV increases rapidly, and the deformation of the membrane gradually becomes stable. Finally, the effect of the membrane material on the flow rate and the deformation of the membrane are studied. The result shows that changes in the material properties of the membrane cause a decrease in the amount of deformation in all stages the all positions of the membrane. This work may provide valuable guidance for the optimization of microfluidic passive valve in microfluidic system.