The purpose of this study was to fabricate poly(epsilon-caprolactone) (PCL)/poly ethylene oxid (PEO)/chitosan (CS) ultrafine fiber in both aligned and random structures using electrospinning technique and their process parameters were optimized. The aligned and random PCL/PEO/chitosan ultrafine fibers were also used as scaffold for tissue engineering and their cell affinity was investigated. In the first part, we inspected the effect of environment conditions, solution properties, process parameters on PCL/PEO/chitosan ultrafine fiber. In the second part, the apparatus of electrospinning to manufacture highly aligned PCL/PEO/chitosan ultrafine fiber was developed. The effects of process parameters such as flow rate, design of collector and rotation speed of collecting drum on the morphology of ultrafine fiber were discussed. In addition, the cross link of PCL/PEO/chitosan ultrafine fiber by cross-linking agent was examined, too. The physical properties, chemical properties, and cell affinities of the aligned PCL/PEO/chitosan ultrafine fiber with or without cross link were measured. The chemical analysis and tensile strength of the ultrafine fiber were characterized using Fourier Transfer Infared Spectrophotometer and Universal Tensile Machine, respectively. The results show that the aligned PCL/PEO/chitosan ultrafine fibrous mat had the capacity to induce cellular alignment and enhance cellular elongation.