Electrospun hybrid chitosan/nylon-6 nanofibrous mats with fiber diameters in the range of 80-310 nm were successfully fabricated using an electrospinning method. Nanofibrous membranes were prepared by nucleophilic reaction of the chitosan's hydroxyl and amidocyanogen with the triazinyl chloride of Cibacron Blue F3GA (CB) ligand. This system was used to study the purification of papain. Physical and chemical properties of the affinity membrane were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), contact angle (CA) and element analysis (EA). The equilibrium adsorption capacity (from Langmuir isotherm data) for papain was 93.46 mg/g affinity membrane. Fifteen layers of the composite affinity membrane were packed into a spin column to separate papain from raw material. Significant amount of the adsorbed papain (about 90.4%) was eluted by 1.0 M NaSCN at pH 9.0, and 4.8-fold purification was achieved in a single step. Experiments on regeneration and dynamic adsorption were also performed. It is shown that this system has the potential to be developed for the industrial purification of the papain.