A thin film composite (TFC) nanofiltration membrane was fabricated through interfacial polymerization on microporous poly(tetrafluoroethylene) (PTFE) substrates, which were fabricated by bi-stretching process (longitudinal and transverse stretching). Multilayers were formed on PTFE substrate via physical twining and chemical linkage. Impacts of bi-stretching ratios on separation properties of TFC nanofiltration membrane were deeply investigated. Results indicate that the increase of longitudinal stretching ratio increased the length of fibrils and induced larger pore sizes and porosities, which resulted in lower values of rejection and higher permeate flux. However, the increase of transverse stretching ratio induced that the pore sizes declined and the porosities increased, which may be due to interlaced fibrils. Accordingly, it induced higher values of rejection and permeate flux. The obtained TFC nanofiltration membrane exhibited good acid stability (over 30 days) and high stability on long term (over 90 days). It could be potentially employed in nanofiltration process.