The changes of skin tissue reflectance spectroscopy before and after being treated with the optical clearing agents of three different types of optical clearing within the wavelength rang of 400～1 000 nm, and the degree of changes in reflectance spectroscopy of each group skin during 0～60 min at 580 nm in vivo were real-time dynamically researched. The reflectance spectroscopy of skin tissue before and after being dealt by the optical clearing agents of glycerol, glucose and propylene glycol was measured using a USB-4000 fiber spectrophotometer at 0, 10, 20, 30, 40, 50 and 60 min. The results showed that the reflectance spectral intensity was distinctly decreased, but the reflectance was significantly increased gradually with the time prolonged. However, different optical clearing agents have different clearing progress. The relative decrease of reflectance of palm skin tissue before and after being dealt by the optical agents of 40% glycerol, 40% glucose and 40% propylene glycol during 10, 20, 30, 40, 50 and 60 min at the wavelength 580 nm is 5%, 7%, 9%, 10%, 11% and 12%, 9%, 13%, 16%, 19%, 21% and 22%, and 14%, 22%, 29%, 32%, 34% and 35%, respectively. The significant improvement in light transmittance and enhancement of light penetration through tissue was demonstrated for all solutions. The effect and processes of optical clearing of skin tissue is not only closely related to the choosing of the clearing agent type, but also related to the treatment time with the skin tissue. The clearing progress of different type optical clearing agent showed the order of 40% propylene glycol, 40% glucose and 40% glycerol.