In order to find a non-invasive way to improve the efficacy of skin optical clearing with topically applied optical clearing agents, the authors researched the changes of reflectance spectroscopy of skin tissues before and after being dealt by low-frequency ultrasound and osmotic active chemical agents within the wavelength range of 400-860 nm, and the degree of changes in reflectance spectroscopy of each group skin during 0-15 min and 15-30 min at 580 nm. The measurements were performed using a AvaSpec-2048 optical fiber spectrometer with integrating-sphere setup. The results of measurements showed that there were a few changes of the reflectance spectroscopy of skin tissues in the control group(Group1)(The skin tissue was dealt with nothing.)during the whole observation; But in the Group 2(The skin tissue was dealt with only low-frequency ultrasound), it was found that the reflectance of the skin tissue showed a significant increase comparing 15 min with 0 min, but the changes in reflectance of the tissues slowly restored the original form following the longer time since the ultrasound stopping; There was also a very fast changes in reflectance of the skins in the Group 3(the skin were dealt with only 80% glycerol)compared with that in the Group 1. The authors also found that there was a very distinct decrease in the reflectance of skin tissues dealt with both low-frequency ultrasound and 80% glycerol group(Group 4), especially during the 0-15 min, and its speed was 4.0 times that of the Group 1 and 2.3 times that of Group 3(During 0-15 min, the reflectance of skin tissues in the Group 1 decreased 1.896%; the reflectance of skin tissue in the Group 3 decreased 3.316%; the reflectance of skin tissues in the Group 4 decreased 7.551%). From the above results, it can be clearly seen that the low-frequency ultrasound and 80% glycerol not only have synergistic effect on optical clearing of skin tissue in vitro, but can change the optical clearing of the skin tissue in a short time.