In this study, we used an epi-illuminated multiphoton microscope to image three main components of ex vivo human tooth. In particular, we obtained two-photon excited autofluorescence (AF) and second-harmonic generation (SHG) images of the enamel, dentin, and periodontal ligaments (PLs) and constructed three-dimensional projections of sequentially and axially acquired images. We found that the enamel has a strong two-photon AF signal, clearly revealing the structures of the enamel rods. The dentin, on the other hand, has both AF and SHG signals. The contrast provided by the combination of these two imaging modalities can be used to reveal the structure of peritubular dentin and to distinguish the less mineralized circumpulpal dentins. The SHG and multiphoton AF imaging also showed the structure of the PL and the distribution of cells around the PL, respectively. For comparison, we also obtained scanning electron microscopy images of the enamel, dentin, circumpulpal dentin, and the PL. Our results demonstrate the effectiveness of using multiphoton microscopy to visualize the major constituents of teeth, including enamel, dentin, and the PL, and the potential of this minimally invasive technique for monitoring the morphological developments during tooth regeneration.