Live cell imaging (LCI) is the approach of non-invasively analyzing dynamic processes in living cells using state-of-the-art microscopy and computer vision techniques. LCI provides exciting and novel insights into cell biology. The present review summarized LCI research in recent years and detailed the role of probes in LCI. We discussed the basic principle and development in LCI technology, including fluorecence resonance energy transfer, fluorescence lifetime imaging microscopy, atomic force microscopy, scanning ion conductance microscopy, confocal microscopy, two-photon excitation fluorescence microscopy and Cell-IQ® System . Then we listed several probes such as green fluorescent protein and quantum dots which are the most widely used probes. Proteins, organelles, nucleotides and other chemicals related to cell biology may all be potential targets. We also discussed the advantages and disadvantages of different probes. Although specific probes in LCI still need to be explored, the application of LCI may be attractive for better understanding of cellular functions.