In this study, an environmentally friendly method for preparation of chitosan crosslinked membranes with different genipin concentrations (0, 125, 250, and 500 mg/L) was developed. Genipin-crosslinked chitosan membranes were used for the immobilization of fungal pectinesterase (PE). PE was efficiently immobilized in chitosan membranes and used for modification of high-methoxylated pectins into low-methoxylated pectins. The charge density (zeta-potential), infrared spectroscopy (FTIR), and ion-exchange chromatography (IEC) revealed the modification of pectin for immobilized PE in genipin-crosslinked chitosan membrane. The zeta-potential results indicated a decrease of -5.2 mV in the pectin control solution, whereas using the PE immobilized in chitosan and genipin-chitosan membranes, the obtained values were -30.45 mV and -36.38 mV, respectively. Genipin-crosslinked chitosan membrane jointly with the immobilized PE represents an eco-friendly support to prepare tailor-made low-methoxylated lime pectin.