The invention discloses a three-dimensional polymer nanofiber membrane with one-dimensional polymer nanofibers orthogonally laminated in a grid shape in quasi-regular arrangement and controlled pore distribution and pore size and a preparation method thereof. A three-dimensional polymer nanofiber membrane with adjustable pore size and porosity can be formed by using an electrospinning pattern forming apparatus, which comprises: the double insulation blocks can make the nano fibers aligned in a specific direction by deforming the electric field; and a current collector rotatable by 90 °. Further, the functional surface coating imparts various properties, and thus can be used for an air filter, a separation membrane, a water treatment filter, a cell culture membrane, and the like.