Abstract. Nanofiber is a widely used biopolymer in medical and industrial applications due to its unique characteristics such as the high surface area to volume ratio and its biocompatibility. This work demonstrates the ability of polyion complex chitosan-alginate to form nanofiber with the addition of polyvinyl alcohol (PVA) by electrospinning. The electrospinning process offers a unique capability to produce natural nanofibers with controlled pore structure using both natural and synthesis polymer or its combination. This research was conducted to optimize the fabrication of chitosan/alginate/PVA nanofibers by varying the conductivity of solution, solvent concentration, and polymer composition. The obtained nanofibers were characterized using Scanning Electron Microscope (SEM) to observe the morphology and Fourier Transform Infrared (FTIR) identify the constituent group. Nanofibers have achieved with the range of mixture solution's conductivity of 0.5-2.0 Mho/cm. The resulted nanofibers showed a uniform nanofiber with average diameter around 200-400 nm. Although higher acetic acid concentration (as a chitosan solvent) did not change conductivity significantly, it resulted in a larger diameter of nanofibers. Higher sodium alginate concentration in the mixture solution produced nanofibers with higher conductivity and larger diameter.