The invention relates to a polylactic acid film. The polylactic acid film comprises an inner layer prepared from a polylactic acid copolymer, at least one middle layer mainly prepared from cellulose and chitin, and at least one surface layer prepared from a polylactic acid copolymer; and based on a weight of the surface layer, the surface layer contains 0.05%-0.6% by weight of crosslinked polymer spherical particles having particle diameters of 2-6 [mu]m, and the middle layer is located between the inner layer and the surface layer. The invention also relates to a preparation method of the polylactic acid film. The preparation method comprises the steps of performing co-extrusion cast compounding on the inner layer, the middle layer and the surface layer, and performing biaxial stretching to obtain the polylactic acid film. According to the multi-layer co-extruded film containing the polylactic acid polymer and the cellulose-chitin prepared by the invention, the friction coefficient is reduced, the adhesion is reduced, the heat sealing property is increased, electrostatic formation and film dust collection are reduced, the film has high biodegradability, the cellulose-chitin layer has a crystal structure through which gas cannot pass, the crystal structure can effectively resist invasion of oxygen, and the prepared film is more suitable as a packaging film for fresh food.