The present study is an attempt to optimize the electrospinning parameters to fabricate chitosan/polyethylene oxide hybrid nanofiber composite with minimum diameter and coefficient of variation (homogeneity) by using response surface methodology (RSM). Based on central composite design (CCD), four factors (the chitosan/PEO ratio, the applied voltage, the needle-to-collector distance, and the spinning angle) was applied to evaluate the individual and combined effects of the parameters on the average diameter of nanofibers. Chitosan (CH) was blended with Polyethylene oxide (PEO) at weight ratios of 1:1, 2:1, 3:1, 4:1, and 5:1. As the composition ratio of chitosan was reduced, the viscosity of the chitosan/PEO blend solution also dropped. The RSM model identified the following conditions as necessary to produce the desired and most uniform CS/PEO fiber diameter: a CS/PEO blend ratio of 2:1 (w/w), a voltage of 25 kV, a distance of 20 cm, and a spinning angle of 45°. The average nanofiber diameter and homogeneity under these conditions were calculated to be 73 nm and 20.5%, respectively, compared to the predicted values of 71 nm and 15.58%, respectively.