The separation of protein plays a critical role in the protein production process. A functionalized nanofiber fabric was prepared by immobilizing Cibacron Blue (CB) on a polyvinyl alcohol (PVA) nanofiber fabric produced by an electrospinning method. To evaluate its separation performance, the adsorption test of bovine serum albumin (BSA), a kind of protein, was conducted using this fabric. The CB molecules were immobilized by the covalent bonding of hydroxyl group of PVA and chlorinated triazine ring of CB under alkaline condition. The adsorption test of CB-enhanced affinity PVA nanofiber fabric was conducted by soaking it into the BSA solution as an adsorbate. As a result, after enhancing affinity by CB, the BSA adsorption amount of the PVA nanofiber fabric indicates a significant increase due to functionalization. Moreover, the adsorption characteristics were heavily dependent on the solution environment.