Single-cell membrane biophysical properties, which including membrane potential, membrane tension, and specific membrane capacitance, can serve as label-free indicators for assessing cell type and state. However, conventional tools limited in their throughput, such as patch clamping and AFM, impose constraints in single-cell membrane biophysical properties detection. The application of microfluidics in single-cell biophysical measurements allows for a convenient and efficient detection with the capability of multi-parameter analysis. In this paper, a microfluidic platform for high-throughput characterization of multiple biophysical properties of single-cell membranes was developed. Using this platform, we successfully measured the membrane relative potential, membrane tension, and specific membrane capacitance of single cells.