Lignin is a biocompatible, biodegradable and renewable biopolymer with macromolecular structure that can be used in production of hydrogel network. The main aim of this research was to electrospin lignin hydrogel in the form of nanofibers by engineering the polymer structure to form stimuli-responsive lignin nanofibers in alkaline pH. For this purpose, a blend of lignin/PVA (polyvinyl alcohol) and citric acid as a friendly cross-linker were electrospun. SEM images showed the best morphology of nanofibers at lignin/PVA of 98:2 with mean diameters of 215 and 176 nm on the static and rotational drum, respectively. The FTIR spectra and X-ray diffraction confirmed the formation of networks between the PVA and lignin chains. The produced nanofibers of lignin can classify as a smart nanohydrogel due to the high swelling ratio in alkaline pH. Antibacterial assays confirmed suitable activities against S. aureus and the fungus Candida albicans for the nanofibers with 20% lignin. Further, the cell cytotoxicity against cell fibroblast of the human skin showed reasonable cell viability. Finally, the nanofibers with 20% lignin collected on the rotational drum indicated the higher strength and modulus of elasticity. In conclusion, the fabricated stimuli-responsive hydrogel with 20% lignin showed suitable performance in antibacterial activity, cytotoxicity and it displayed good mechanical properties. In addition, due to the repulsive interactions between ionized carboxylic groups under alkaline pH, the swelling occurs. As a result, these nanofibers are potentially applicable in biomedical applications such as wound dressing.