This study aimed to investigate the significance of the physico-chemical environment during trypsin adsorption onto a highly bio-specific affinity membrane. The affinity matrix polysulfone (PSf) membranes were prepared via a simple dry/wet phase inversion technique. Surface modification of PSf membranes was employed using chitosan in order to improve membrane hydrophilicity. Glutaraldehyde and ovomucoid were used as the membrane activator and affinity ligand, respectively. Inspection of membrane morphology was done using scanning electron microscopy. The functional groups on the membrane surface were determined using Fourier-transform infrared spectroscopy equipped with attenuated total reflection (ATR-FTIR). In order to determine the optimum conditions for the maximum adsorption capacity of trypsin, adsorption studies were performed at different pH levels (5, 7, 8, 10, 12), ionic strengths (0.01, 0.05, 0.1, 0.3 and 0.5 M) and initial trypsin concentrations (0.1, 0.3, 0.5, 0.7 and 0.9 mg/ml). The optimum adsorption was obtained with a 0.9 mg/ml initial trypsin solution at pH 7 and an ionic strength of 0.1 M.