Unlike the ensemble-averaging measurements, the single-molecule imaging and tracking (SMIT) in living cells provides the real-time quantitative information about the locations, kinetics, dynamics and interactions of individual molecules in their native environments with high spatiotemporal resolution and minimal perturbation. The past decade has witnessed a transforming development in the methods of SMIT with living cells, including fluorescent probes, labeling strategies, fluorescence microscopy, and detection and tracking algorithms. In this review, we will discuss these aspects with a particular focus on their recent advancements. We will then describe representative single-molecule studies to illustrate how the single-molecule approaches can be applied to monitor biomolecular interaction/reaction dynamics, and extract the molecular mechanistic information for different cellular systems.