Currently, tissue optical clearing technique has shown a great potential in enhancing the capabilities of non-invasive light-based diagnostic and imaging techniques due to the increased light penetration into tissue. In order to facilitate the clinical availability of tissue optical clearing technique by the use of hyperosmotic agents, this study introduces oleic acid, a mono-unsaturated fatty acid which is generally believed to be safe, as enhancer and investigates the synergistic effect of oleic acid and propylene glycol (PG) on optical clearing of skin tissue in vitro. Experimental results from near infrared spectroscopy, mass loss measurement and transdermal skin resistance (TSR) assessment showed that, compared with dimethyl sulfoxide (DMSO) as enhancer, oleic acid obtained the similar clearing effect. However, due to its potential toxicity, DMSO has been controversial in clinical application. Therefore, in terms of optical application and clinic safety, the results presented revealed that oleic acid could be an optimum choice as enhancer for optical clearing of skin tissue.