The invention relates to a preparation method of biomass-based nano-silica aerogel. The method comprises the following steps: acidifying straws used as a raw material by hydrochloric acid to remove impurities, pyrolyzing the acidified straws at a high temperature, and combing a high-speed vibration ball mill with an ultrasonic dispersion process to extract biomass-based nano-silica from the raw material straws; carrying out a heating reaction on the biomass-based nano-silica and a NaOH solution, and cooling and filtering the obtained solution to obtain a biomass-based water glass solution; adding sulfuric acid to adjust the pH value to 1-2, and dropwise slowly adding the NaOH solution to make the pH value reach 7-8, carrying out continuous stirring standing, and cleaning the obtained material with deionized water to remove impurities in order to synthesize biomass-based hydrogel; exchanging the hydrogel with an anhydrous ethanol solvent; and freeze-drying the exchanged biomass-based hydrogel by using a vacuum freeze drying machine to obtain the biomass-based nano-silica aerogel. The obtained nano-silica aerogel has the advantages of high specific surface area, reasonable pore structure, low apparent density and excellent thermal performance, can be used in the fields of buildings, medicines and chemical engineering catalysis materials, and realizes waste recycling.