Single-cell profiling is key to uncover the cellular heterogeneity and drives deep understanding of cell fate. In recent years, microfluidics has become an ideal tool for single-cell profiling owing to its benefits of high throughput and automation. Among various microfluidic platforms, microwell has the advantages of simple operation and easy integration with in situ analysis ability, making it an ideal technique for single-cell studies. Herein, recent advances of single-cell analysis based on microwell array chips are summarized. We first introduce the design and preparation of different microwell chips. Then microwell-based cell capture and lysis strategies are discussed. We finally focus on advanced microwell-based analysis of single-cell proteins, nucleic acids, and metabolites. The challenges and opportunities for the development of microwell-based single-cell analysis are also presented.