Polyamide reverse osmosis (RO) membranes possessing high water permeance and high salt rejection are highly beneficial for efficient desalination and wastewater treatment. However, achieving desired membrane properties toward both fast water transport and outstanding salt rejection remains a great challenge. Herein, we report the fabrication of high-performance polyamide RO membrane via interfacial polymerization (IP) on a heterogeneous substrate featuring a hydrophobic surface and polyaniline modified hydrophilic pores. The heterogeneous substrate, combining the advantages of conventional hydrophilic and hydrophobic substrates for IP process, leads to the formation of an ultra-thin polyamide film with rough morphology. The obtained RO membranes thereby not only have simultaneously enhanced water permeance (3.28 L m−2h−1 bar−1) and salt rejection (NaCl rejection 99.5 %), but also exhibit outstanding pH stability, anti-fouling performance, and long-term stability. In addition, the regulation mechanism of the heterogeneous substrate on IP was comprehensively investigated by combing molecular simulation and experiments. Our work provides a novel design direction of substrate used for fabricating RO membrane with outstanding performance.