Fluorescence proteins (FPs) have been widely used for live-cell imaging in the past decade. This review summarizes the recent advances in FP development and imaging technologies using FPs to monitor molecular localization and activities and gene expressions in live cells. We also discuss the utilization of FPs to develop molecular biosensors and the principles and application of advanced technologies such as fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), and chromophore-assisted light inactivation (CALI). We present examples of the application of FPs and biosensors to visualize mechanotransduction events with high spatiotemporal resolutions in live cells. These live-cell imaging technologies, which represent a frontier area in biomedical engineering, can shed new light on the mechanisms regulating mechanobiology at cellular and molecular levels in normal and pathophysiological conditions.