The invention discloses a preparation method of a chitosan derivative aquagel, belonging to the field of biological medical materials. The preparation method of the chitosan derivative aquagel comprises the following steps: carrying out alkalification, swelling, chloroethanol modification, after-treatment and other series technical processes on chitosan to prepare hydroxyethyl chitosan, carrying out glycidyl methacrylate graft modification, and finally, carrying out photochemical polymerization to form the aquagel. The obtained aquagel has the advantages of favorable biocompatibility, low toxicity, short gelling time, controllable and adjustable crosslinking density, favorable elasticity, high structural stability and the like. The preparation method has the characteristics of accessible raw materials, simple technique, mild reaction conditions and the like. The obtained aquagel can be used in the aspects of cell delivery, tissue engineering, cell three-dimensional culture and the like.