Fluorescence-based molecular imaging has long and widely been used in the field of biomedical research, and now has become an indispensable tool. The development of confocal microscopy in the 1980s was a major breakthrough in the field. It can optically slice biological samples along the optical axis, which had been impossible with the conventional fluorescent microscopy. In this review, we first discuss the basic principle, and advantages of the confocal microscopy, and next describe the recent topics on the improved microscopy including high-speed confocal microscopy, FRET(fluorescence resonance energy transfer), FRAP (fluorescence recovery after photobleaching), and two photon excitation microscopy. Finally, we illustrate some examples of the biomedical applications of the confocal microscopy.