The invention relates to a method and system for processing a nano-cone array on a silk film by utilizing femtosecond laser, and belongs to the technical field of femtosecond laser application. The method comprises the steps that firstly, the silk film is prepared through a chemical method, then a single femtosecond laser pulse is focused on the surface of the silk film through a microscope objective lens, and a nano-cone structure with the diameter of 200-500 nm and the height of 17-170 nm is obtained by regulating the energy flux incident on the surface of the silk film. According to the method and the system, under the irradiation of the femtosecond laser pulse sequence, the silk film is continuously moved through an electronic control translation stage, and therefore the preparation ofthe nano-cone array with the pitch of 600-1500 nm is realized. The method is based on the multi-photon absorption expansion effect of silk fibroin on the femtosecond laser, and therefore the nano-cone array far smaller than the diffraction limit is processed on the surface of the silk film. The system and the method have the advantages of being high in precision, high in flexibility, contactless,maskless and capable of being conducted in an atmospheric environment, and a feasible method is provided for preparing a biological photoelectric device.