The newly emerging field in organic electronics is to control the molecule-substrate interface properties at a two-dimensional (2D) limit via interfacial interactions, which paves the way for driving the molecular assembly for highly ordered 2D molecular crystalline films with precise molecular layers and large-area uniformity. Here, by exploiting molecule-substrate van der Waals (vdW) interactions, we demonstrate thermally induced self-assembly of 2D organic crystalline films exhibiting well-defined molecular layer number over a millimeter-sized area. The organic field-effect transistors (OFETs) with bilayer films show excellent electrical performance with a maximum mobility of 12.8 cm2 V-1 s-1. Moreover, we find that the monolayer films can act as interfacial molecular templates to construct heterojunctions with well-balanced ambipolar transport behaviors. The capability of thermally induced self-assembly of 2D molecular crystalline films with controllable molecular layers and scale-up coverage opens up a way for realizing complicated electronic applications, such as lateral heterojunctions and superlattices.