Accurate analysis of the mechanical, electrical, and optical properties of single cells can provide useful information on their physiological and pathological states. Microfluidics has become a promising tool for single-cell analysis because it greatly reduces the operation complexity, reduces the reagent consumption, and enables the integration of multiple functions. In recent years, the rapid development of microfluidic detection technologies is laying a foundation for single-cell analysis, which promotes the in-depth analysis and understanding of cells. In this review, we provide a comprehensive overview of the most recent advances on label-free microfluidics for analyzing the biophysical properties of single cells. Herein, we present the applications of cell biophysical properties for identifying cell types, distinguishing between healthy and diseased cells, and studying the drug resistance of cells. Lastly, the advantages, limitations, and technical characteristics of each method are discussed.