The invention discloses a preparation method of a one-way wet conduction nano-fiber multilayer composite membrane with the wettability gradient. The method comprises the following steps that hydrophilic nano materials are dispersed in solvent, the nano materials are dispersed uniformly through ultrasound, a hydrophilic polymer is dissolved in the dispersing agent, a spinning solution A1 is obtained, and a hydrophilic nano-fiber membrane is deposited on a receiving base material through an electrostatic spinning method; hydrophilic nano materials are dispersed in solvent, the nano materials are dispersed uniformly through the ultrasound, a hydrophilic polymer is dissolved in the dispersing agent, and a spinning solution A2 is obtained; a hydrophobic polymer is dissolved in solvent, and a spinning solution B1 is obtained; the two spinning solutions are deposited on the hydrophilic nano-fiber membrane to form at least one diversion layer through the electrostatic spinning method; a hydrophobic polymer is dissolved in solvent, a spinning solution B2 is obtained, a hydrophobic nano-fiber membrane is deposited on the diversion layers through the electrostatic spinning method, and the one-way wet conduction nano-fiber multilayer composite membrane with the wettability gradient is obtained.