The separation performance of current organic solvent nanofiltration (OSN) membranes are still not satisfied. In this work, an ultra-thin TpHz covalent organic frameworks (COFs) interlayer was in situ constructed in advance on polyimide substrate, then an ultra-thin polyamide separation layer incorporated with amino-functionalized graphene quantum dots (af-GQDs) was constructed via interfacial polymerization (IP). A high performance interlayered thin-film nanocomposite (iTFN) OSN membrane with integrally covalent bonding structure, ultra-thin and ultra-smooth surface was obtained after post crosslinking and activation. The effect of ultra-low concentrations for the polyamide reaction monomers, the COFs monomer Tp and the doped af-GQDs nanoparticle reveals that they could help to form a very thin (7–8 nm in thickness) and defect-free skin layer with very smooth surface (about 2.7 nm in roughness). The iTFN OSN membrane shows excellent solvent permeance and solute selectivity, with an ethanol permeance reaching 97.6 L m−2 h−1 MPa−1 and an RDB rejection higher than 99%. After a 180 h long-term filtration with 100 mg L−1 RB/DMF solution, it remained an RB rejection of higher than 99%, demonstrating its superior resistance to strong polar solvent. Our work paves a unique way for the fabrication of novel OSN membrane with both in situ constructed nanomaterials interlayer and nanomaterials incorporated in skin layer.