The invention provides a cell transfection device, a cell transfection method and a micro-channel manufacturing method. The cell transfection device comprises a micro-fluidic chip and a tunable extrusion mechanism, a micro-channel is arranged in the micro-fluidic chip, a limited space is arranged in the micro-channel, and a plurality of allogenic materials are arranged in the micro-channel; the tunable extrusion mechanism comprises a micro needle tip, a piezoelectric actuator and a cantilever beam; and when the piezoelectric actuator vibrates, the micro needle tip is driven to move up and down, extrudes or is separated from the micro-fluidic chip, cell membranes of cells in the limited space are driven to be damaged to generate through holes, and the allogenic materials enter the cells through the through holes. According to the cell transfection device, the cell transfection method and the micro-channel manufacturing method, the micro-fluidic chip is regularly extruded or separated through the tunable extrusion mechanism, so that the width of the micro-channel is variable, the micro-channel is suitable for cells with different sizes, and the problem that a traditional micro-channel with a fixed width is only suitable for cells with specific sizes or deformation force and has low transfection efficiency on macromolecular nano allogenic materials is solved.