A preparation method of mesoporous carbon-based solid acid includes the steps of dissolving water-soluble carbohydrate in water to obtain a precursor solution, dissolving nonionic surface active agent in the solution to obtain a mixed solution of a precursor and a template, adding inorganic acid to transfer the solution into a culture dish, evaporating the culture dish at a temperature of 40-60 DEG C for 6 hours, conducting thermal setting at a temperature of 100-150 DEG C for 6-24 hours to obtain a mixture of the precursor and the template, and directly calcinating the obtained mixture under the inert atmosphere to remove the template to obtain the mesoporous carbon-based solid acid. Carbohydrate low in price is adopted as the precursor, inorganic acid serves as carburizer, the acid is prepared through an evaporation, induction, self-assembly and carbonization method, the method is easy to operate and beneficial to large-scale production, and the mesoporous carbon-based solid acid synthesized through the method has a large specific surface and abundant surface acid groups and can be widely applied to heterogeneous acid catalysis and biomass conversion.