This paper investigates the basic deflection properties of polydimethylsiloxane (PDMS) membrane as an actuator component in a microfluidic device. Polydimethylsiloxane membrane is a widely used structure in various applications in microfluidics. Most of the applications using PDMS membrane as actuators are pumps, valves, microlenses, and cell stimulators. In these applications, PDMS membranes are deflected to function by applied pressure. However, based on our literature survey, correlations between thickness, applied air pressure, and the deflection properties of replaceable PDMS membrane have not been theoretically and experimentally investigated yet. In this paper, we first conducted a simulation to analyze the relationship between deflection of the replaceable PDMS membrane and applied pressure. Then we verified the deflection of the PDMS membrane in different experimental conditions. Finally, we demonstrated that the PDMS membrane functioned as a valve actuator in a cell-capturing device as one application. We expect this study would work as an important reference for research investigations that use PDMS membrane as an actuator.