Abstract: To solve the problem of the single thickness of the light-sheet fluorescence microscopy, the design of the variable light-sheet illumination system was carried out based on zoom beam expanding. First, each group of the light-sheet illumination system was calculated using Gaussian optics to obtain the relation between the thickness of the light-sheet and the expansion ratio as well as the relation among the expansion ratio, the lateral magnification and focal length of each group. Then a variable light-sheet illumination system based on 10beam expanding was designed to generate the light-sheet with continuously variable thickness and length. Finally, the parameters and uniformity of the light-sheet and the system tolerance were analyzed. The results show that the thickness of the continuously variable light-sheet is 3.33 m to 33.3 m, and the illumination uniformity is 0.65, 0.4 and 0.61 on the YOZ plane within 60% light-sheet height at low (1), medium (6) and high (10) expansion ratios, respectively. Tolerance analysis shows that the maximum change of the light-sheet thickness is less than the 15% of design value at expansion ratio of 1, less than 6% at 6and 10. The design realizes the continuous variation of the light sheet thickness, and the region of 60% light-sheet height is favorable for the observation of the sample.