The invention discloses a preparation method of a ramie bone cellulose nanocrystalline self-assembled structural color thin film. The preparation method comprises the following steps: firstly, mixing ramie bone meal with water, and performing steam-explosion treatment; secondly, mixing the pretreated ramie bone meal with a sodium hydroxide solution and cooking to obtain ramie bone coarse cellulose; mixing the ramie bone coarse cellulose with hydrogen peroxide; performing oxidizing reaction under the condition of ice bath to obtain ramie bone cellulose; mixing the ramie bone cellulose with a sulfuric acid solution and hydrolyzing to obtain a reaction solution containing ramie bone cellulose nanocrystalline; centrifuging and dialyzing the reaction solution to obtain a purified nanocrystalline cellulose suspension; removing solvent in the suspension to obtain a self-assembled nanocrystalline cellulose thin film. The preparation method disclosed by the invention is simple; in addition, the size of the cellulose nanocrystalline is controllable; the obtained cellulose nanocrystalline thin film can be applied to the field of biomedical and polymer materials.