Microchips are fundamental tools for single-cell analysis. Although various microfluidic methods have been developed for single-cell trapping and analysis, most microchips cannot trap single cells deterministically for further analysis. In this paper, we describe a novel resistance-based microfluidic chip to implement deterministic single-cell trapping followed by immunofluorescence staining based on the least flow resistance principle. The design of a large circular structure before the constriction and the serpentine structure of the main channel made the flow resistance of the main channel higher than that of the trapping channel. Since cells preferred to follow paths with lower flow resistance, this design directed cells into the capture sites and improved single-cell trapping efficiency. We optimized the geometric parameters using numerical simulations. Experiments using A549 and K562 cell lines demonstrated the capability of our chip with (82.7 ± 2.4)% and (84 ± 3.3)% single-cell trapping efficiency, respectively. In addition, cells were immobilized at capture sites by applying the pulling forces at the outlet, which reduced the cell movement and loss and facilitated tracking of the cell in real time during the multistep immunofluorescence staining procedure. Due to the simple operation, high-efficiency single-cell trapping and lower cell loss, the proposed chip is expected to be a potential analytical platform for single tumor cell heterogeneity studies and clinical diagnosis.