Single-cell analysis is an effective method for conducting comprehensive heterogeneity studies ranging from cell phenotype to gene expression. The ability to arrange different cells in a predetermined pattern at single-cell resolution has a wide range of applications in cell-based analysis and plays an important role in facilitating interdisciplinary research by researchers in various fields. Most existing microfluidic microwell chips is a simple and straightforward method, which typically use small-sized microwells to accommodate single cells. However, this method imposes certain limitations on cells of various sizes, and the single-cell capture efficiency is relatively low without the assistance of external forces. Moreover, the microwells limit the spatiotemporal resolution of reagent replacement, as well as cell-to-cell communication. In this study, we propose a new strategy to prepare a single-cell array on a planar microchannel based on microfluidic flip microwells chip platform with large apertures (50 μm), shallow channels (50 μm), and deep microwells (50 μm). The combination of three configuration characteristics contributes to multi-cell trapping and a single-cell array within microwells, while the subsequent chip flipping accomplishes the transfer of the single-cell array to the opposite planar microchannel for cells adherence and growth. Further assisted by protein coating of bovine serum albumin and fibronectin on different layers, the single-cell capture efficiency in microwells is achieved at 92.1% ± 1%, while ultimately 85% ± 3.4% on planar microchannel. To verify the microfluidic flip microwells chip platform, the real-time and heterogeneous study of calcium release and apoptosis behaviours of single cells is carried out. To our knowledge, this is the first time that high-efficiency single-cell acquisition has been accomplished using a circular-well chip design that combines shallow channel, large aperture and deep microwell together. The chip is effective in avoiding the shearing force of high flow rates on cells, and the large apertures better allows cells to sedimentation. Therefore, this strategy owns the advantages of easy preparation and user-friendliness, which is especially valuable for researchers from different fields.