The invention discloses hyaluronic acid derivative hydrogel and a preparation method thereof and belongs to the field of biomedical materials. The preparation method includes the steps that methacrylic acid 2-amino ethyl ester and L-lysine are grafted on hyaluronic acid molecules through a carbodiimide method, then an obtained hyaluronic acid derivative and a water-soluble photoinitiator are subjected to photochemical polymerization in water, and then the hyaluronic acid derivative hydrogel is obtained. The hyaluronic acid derivative hydrogel is stable in mechanical performance, high in elasticity, uniform in hole diameter, simple in process, mild in reaction condition and short in gelation time, and a product has syringeability. The hyaluronic acid derivative hydrogel can be used as a 3D tumor model matrix, a tissue engineering scaffold material, a drug carrier, a wound dressing and the like.