Enzyme immobilization onto nanofiber matrices presents a promising avenue for various biotechnological applications. In this study, the electrospinning method was used to produce nanofibers composed of polyvinyl alcohol (PVA), beta cyclodextrin (βCD), and collagen, subsequently investigating the immobilization of arginase onto this characterized carrier matrix. The optimal production parameters for the collagen-blended nanofibers were determined as follows: a PVA concentration of 6% (w/v), a βCD concentration of 2% (v/v), a collagen concentration of 1% (w/v), an electrical voltage of 13 kV, a needle-collector distance of 15 cm, and an injection speed of 0.5 mL/h. The structural and morphological properties of the nanofibers were evaluated through comprehensive analyses including ATR-FTIR, TGA, and SEM techniques. Following this, arginase immobilization onto the collagen-blended nanofibers was accomplished through adsorption and cross-linking techniques. At high temperatures, the free arginase lost 90% of its activity after 1 h, while the immobilized arginase retained about 90% of its activity under the same circumstances. The arginase immobilized collagen-blended nanofiber retained 50% of its activity after 29 reuses. Moreover, the nanofibers enhanced enzyme stability properties, including storage capacity, thermal and pH stability. As a result, this study introduces an innovative strategy utilizing collagen-blended nanofiber matrices for enzyme immobilization, highlighting their potential applications in biocatalysis and enzymatic process.