The main objective of this research is to produce high performance nanofiltration (NF) hollow fiber membranes for Cyclodextrin Glycosyltransferase (CGTase) separation. For the first stage of this study, three types of spinning solution had been formulated by using titration method. These spinning solutions were formulated close to its cloud point (binodal line) in order to speed up the coagulation of nascent fibers so that the relaxation effect of molecular orientation was reduced. The dry/wet spinning process was applied in hollow fiber fabrication with water as bore fluid. At the second stage, hollow fibers were fabricated at four different type of dope extrusion rate (DER) ranging from 2.0 to 3.5 cm3/min. It was found that the optimum condition occurred at DER 2.5 cm3/min, which yields an optimal performance of sodium chloride rejection and better membrane morphology. The results exhibited that as the DER increased, the rejection value increases until critical level is achieved but the flux value gradually reduced. The optimized DER, at 2.5 cm3/min was selected to fabricate the fibers at different air gap length. Thus, by increasing air gap length, the rejection value is significantly increased. The results suggested that it is possible to separate the CGTase enzyme up to 99.11% with the flux of about 0.47 l/m2.h and enhance the NF hollow fiber membranes performance in CGTase separation by the approach proposed in this study. Finally, at the last stage, the produced NF membranes were characterized by using Scanning Electron Microscope (SEM) and Attenuated Total Reflection Fourier Transform Infrared (FTIR-ATR). Results revealed that phase inversion and rheological factors significantly influencing the separation performance of Polyethersulfone (PES) NF hollow fiber membranes
