We investigated the effect of thiazone, a widely used penetration enhancer, on in vitro porcine skin and muscle tissue by single-integrating sphere technique during optical clearing (OC) treatment. The results showed that thiazone induced an increase on the total transmittance of skin which led to a reduction in that of muscle in the spectral range from 400 to 800 nm. Small particles crystalized out from the thiazone-treated muscle were observed by microscopy imaging. With the help of x-ray diffraction measurement, we ascertained that the crystal was a single-crystal of thiazone, which mainly induced an increase of the scattering. Contrast transmittance measurements carried on the mixture of water, thizaone–propylene glycol solution showed that the free water in muscle could be the main reason for the thiazone crystallization. Therefore, during OC treatment of thiazone, the remarkable effect on skin and the noticeable inhibition effect on subcutaneous muscle tissue after penetrating into the skin should be considered. The experimental results provide such a reference for the choice of penetration enhancer.