The invention discloses a preparation method of a nanofiber non-woven fabric. A thermoplastic polymer and a substrate which have thermodynamic incompatibility performance are subjected to melt blending according to a certain mass ratio, through melt extrusion and melt blowing, composite fibers are obtained, and the single-layer composite fibers are deposited on a receiving device; under the adhesion effect of the composite fibers, a single-layer composite fiber web is obtained; the substrate in the composite fiber web is dissolved and removed by adopting a solvent, a nanofiber web with a thermoplastic polymer phase is obtained, then the same method is adopted for collecting the second polymer nanofiber web layer, the third polymer nanofiber web layer,..., and the n polymer nanofiber web layer on a single-layer nanofiber non-woven fabric in sequence, and finally, the nanofiber non-woven fabric is obtained through hot rolling reinforcement treatment. By means of the preparation method, the nanofiber non-woven fabric with the nanofiber diameter smaller than 200 nanometers and the thickness of 0.2-5 microns can be prepared, and the preparation method of the nanofiber non-woven fabric has the advantages that the fiber diameter uniformity and strength are high, the preparation method is simple, the cost is low, and large-scale preparation can be achieved.