Investigation of cell behavior under different environments and manual operations can give information in specific cellular processes. Among all cell-based analysis, single-cell study occupies a peculiar position, while it can avoid the interaction effect within cell groups and provide more precise information. Microfluidic devices have played an increasingly important role in the field of single-cell study owing to their advantages: high efficiency, easy operation, and low cost. In this review, the applications of polymer-based microfluidics on cell manipulation, cell treatment, and cell analysis at single-cell level are detailed summarized. Moreover, three mainly types of manufacturing methods, i.e., replication, photodefining, and soft lithography methods for polymer-based microfluidics are also discussed.