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The effect of optical cleaning method combined with laser speckle imaging (LSI) was discussed to improve the detection depth of LSI due to high scattering characteristics of skin, which limit its clinical application. A double-layer skin tissue model embedded with a single blood vessel was established, and the Monte Carlo method was used to simulate photon propagation under the action of light-permeating agent. 808 nm semiconductor and 632.8nm He–Ne lasers were selected to study the effect of optical clearing agents (OCAs) on photon deposition in tissues. Results show that the photon energy deposition density in the epidermis increases with the amount of tissue fluid replaced by OCA. Compared with glucose solution, polyethylene glycol 400 (PEG 400) and glycerol can considerably increase the average penetration depth of photons in the skin tissue, thereby raising the sampling depth of the LSI. After the action of glycerol, PEG 400, and glucose, the average photon penetration depth is increased by 51.78%, 51.06%, and 21.51% for 808 nm, 68.93%, 67.94%, and 26.67% for 632.8 nm lasers, respectively. In vivo experiment by dorsal skin chamber proves that glycerol can cause a substantial decrease in blood flow rate, whereas PEG 400 can significantly improve the capability of light penetration without affecting blood velocity, which exhibits considerable potential in the monitoring of blood flow in skin tissues.