Abstract: This study aimed to obtain a lentinan-specific fluorescent probe for the in situ visualization of lentinan. A carbohydrate-binding module with potential lentinan-binding capacity was discovered in a β-1,3-D-glucanase by using the bioinformatics techniques, and further recombinantly expressed. The binding specificity of the expressed protein, which was named as LBM was determined by the microtiter plate assays. The results indicated that LBM displayed a desired specificity for lentinan. The protein exhibited positive binding signals to lentinan, while could not bind to several examined polysaccharides including barley β-glucan, curdlan, or konjac glucomannan. Furthermore, the first lentinan-specific fluorescent probe was successfully constructed by fusing LBM with a green fluorescent protein EmGFP, based on which the in situ visualization of lentinan in Lentinula edodes was realized. It was shown that lentinan was not only presented in the mycelial cell wall, but also amorphously distributed in the interstitial space and inside the cell. The construction of the EmGFP-LBM provided a promising tool for the in situ visualization of lentinan.