Intracellular bioactive molecules and their dynamic interactions play a crucial role in maintaining physiological homeostasis, in addition to facilitating pathological progression. Therefore, elucidation of their interactions and cross-talking is of great significant in acquiring a deep understanding of various pathophysiological processes. Fluorescent probes have been demonstrated to be efficient imaging tools for the in situ visualization of molecular events. However, it is still a major challenge to visualize the dynamic intermolecular interactions within living cells. This chapter focuses on recent advances in the fluorescence imaging of dynamic interactions of bioactive molecules in live cells, especially the design of fluorescent probes capable of the simultaneous detection of multiple biomolecules and their biological applications in cell models. These issues open up a new field of research and also great challenges in chemical biology.