We report a fabrication method of polyamide (PA) thin film composite reverse osmosis membranes, so-called support-free interfacial polymerization (SFIP). In contrast to conventional interfacial polymerization (IP) where a PA layer is formed in-situ on top of a support, in this SFIP method the PA layer is first formed at the interface without a support, followed by attachment onto a support. Enhancing the chemical adhesion between the PA layer and a polyacrylonitrile support through the chemical modification on the support leads to the fabrication of defect-free membranes which outperform the conventional IP-assembled membranes. Importantly, SFIP allows for the precise characterization of the PA layer and the PA-support interface by easily isolating each membrane component. SFIP produces a thinner and smoother PA structure with a more wettable and less negatively charged surface than its IP-assembled counterparts, presumably due to uniform and promoted amine diffusion during film formation. Furthermore, it was found that the bottom surface of the SFIP-assembled PA has a porous structure with higher hydrophilicity and a marginally lower negative charge than its opposite surface. The SFIP method provides a versatile platform to study the fundamental membrane structure-performance relationship and to develop high performance membranes.