A fatal problem faced by conventional nanofiltration (NF) membranes is that they cannot be dry-stored without protective agent. Hereon, an anti-drying NF membrane is successfully constructed on a PTFE microfiltration (MF) substrate. It is demonstrated that the selection of PTFE MF substrate is the key factor for ensuring the desiccation tolerance. Meanwhile, a novel interfacial polymerization strategy denotes (one and a half)-step interfacial polymerization (3/2-IP) makes it possible to construct a defect-free polyamide film on the PTFE substrate. During the 3/2-IP process, IP reaction is doubly proceeded only by soaking piperazine (PIP) solution once and trimesoyl chloride (TMC) solution twice, because PIP droplets will be reproduced on membrane surface due to the interfacial tension, vapor condensation and gas driven during heat-treatment. The first IP reduces the substrate pore size and improves its hydrophilicity. Then an intact polyamide film with nano-wrinkled structure is produced in the second IP reaction. Our findings provide a valuable route for fabricating thin-film composite (TFC) NF membranes with desiccation tolerance and desirable separation performance.