Although Janus membranes have been fabricated for the separation of oil/water mixtures, simplifying the construction processes is still a thorny challenge. In this work, a facile and easily scale-up approach based on in situ mussel-inspired chemistry is reported for the first time to prepare the Janus membrane with unidirectional oil/water separation. Here, the poly (vinylidene fluoride)/polydopamine/polyethyleni mine (PVDF/PDA/PEI) membranes on the polyester non-woven fabric were fabricated via non-solvent induced phase separation (NIPS) technique. In the coagulation bath, the reactive oxygen species (ROS) produced by CuSO4/H2O2 improved the formation of the hydrophilic PDA/PEI on the top surface of the membrane. The hydrophobic bottom surface was received after the membrane was stripped from the supporting fabric. Scanning electronic microscopy (SEM), confocal laser scanning microscopy (CLSM), attenuated total reflectance Fourier transform infrared spectrometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive spectrometer (EDS) were used to characterize the membrane. The Janus membrane showed asymmetric wettability with 152.2 degrees underwater oil contact angle of F-T5 and 153.3 degrees underoil water contact angle of B-T5. Furthermore, by this in situ mussel-inspired chemistry, the Janus membrane showed excellent water and oil separation, antifouling performance and stability, which can potentially be used for unidirectional oil/water separation.