In this study, Polycaprolactone (PCL)-based nanofibers containing protein-based egg albumin (EA) were produced via electrospinning. Before the electrospinning process, solutions containing EA at different concentrations were prepared, and the conductivity, viscosity, and surface tension values of these solutions were determined. PCL was used as a carrier polymer in the solutions, and the PCL concentration was kept constant at 7 wt%. EA was present in all six solutions at concentrations of 0, 1, 3, 5, 7, and 9 wt%, respectively. All nanofibers were characterized morphologically, chemically, and thermally. The viscosity, conductivity, and surface tension of the solutions increased with the concentration increasement of EA. Afterward, the electrospinning process was achieved under optimum process parameters and ambient conditions. Uniform, smooth, and homogeneous nanofibers were produced via electrospinning. The Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FT-IR), X-ray Diffraction (XRD), Thermogravimetric Analysis (TGA), and Differential Scanning Calorimetry (DSC) analysis were performed as part of characterization studies. Besides, in-vitro EA release behavior from nanofibers was determined via UV–vis spectrophotometry. When the SEM images were examined, the particles of EA were seen on the web surface. According to the results of chemical and thermal analysis, the presence of PCL and EA was proven in the produced nano web. XRD analysis revealed a decrease in the crystallinity of the protein-based nanofiber with the addition of EA. According to in-vitro analysis results, it was determined that EA showed immediate release from PCL-based nanofibers. In-vitro cytotoxicity test on L929 fibroblasts via MTT assay showed that the prepared nanofibers were not toxic to the cells.