The invention relates to a well-ordered modified nanofiber membrane and preparation and application thereof. The nanofiber membrane is a polycaprolactone modified collagen/silk fibroin fiber membrane, and the pore diameter is 10-25 micrometers. A preparation method comprises the steps that collagen, silk fibroin and polycaprolactone are dissolved in a solvent and stirred and mixed to be uniform, and a spinning solution is obtained; under the room temperature condition, electrostatic spinning is conducted through the spinning solution, drying is conducted, crosslinking is conducted, water washing and vacuum drying are conducted, and the well-ordered modified nanofiber membrane is obtained. The application in serving as a cell culture shelf is achieved. According to the well-ordered modified nanofiber membrane and the preparation and application thereof, the method is simple, operation is convenient and rapid, the fiber membrane of which the fibers are well ordered is obtained through a blending modification and changing collector, the porosity of the fiber membrane is increased, the mechanical property and biocompatibility are good, cells can enter the shelf to grow conveniently, and a good application prospect in cell culture is achieved.