The relatively poor selectivity of electrospun nanofiber membrane (ENM) substrate supported thin film composite forward osmosis (eTFC-FO) membranes has hindered their applications. In this study, to overcome the high roughness and large pore size of ENM substrate, a smooth and hydrophilic polydopamine (PDA) interlayer was fabricated on a polyvinylidene fluoride (PVDF) ENM, which was found to be favourable for the formation of a thinner, more cross-linked and less defective polyamide (PA) active layer on the ENM substrate. Desalination tests proved the great effectiveness of PDA interlayer on performance improvement for the eTFC-FO membrane. A quite high water flux (43.0 LMH) and ions rejection (>97.0%) were achieved for a real desulfurization wastewater desalination tested in FO mode. The alleviated PA defects by the PDA interlayer could greatly improve the selectivity. As a comparison with PDA interlayer, PDA coating on each individual nanofiber inside the PVDF ENM substrate was also prepared for eTFC-FO membrane, which would not overcome the disadvantages of rough and large pore sized ENM substrate for PA preparation. Mechanisms of PA formation and performance improvement were detailly discussed in this work.