Optical coherence tomography (OCT) is used to investigate the microstructure changes of human fingernail induced by hydration. Images of nail plate are obtained to display the morphology of fingernail and to disclose the keratinization of basal cells of nail plate. Combined with digital vernier caliper, this imaging technology is used to evaluate thicknesses and changes of nail in vitro after immersion with time. OCT images of nails show that the dorsal and ventral layers of nails have similar thicknesses which are much thinner than intermediate layer. The total thickness of fingernail exponentially increases with immersion time, and the saturating phenomenon appears at about 12 min. Three layers show different contributions to the total increase of the thickness of 17.4%. Microstructure changes in vivo are similar to the results in vitro. The changes of optical path length also could be evaluated by this method. OCT is capable of reflecting precise microstructure changes, and it has the potential to provide physician with a modern and objective diagnostic standard for nail inspection (NI) and to monitor disorders in Chinese traditional medicine (CTM) clinical practice and research.