A biocompatible and biodegradable hybrid chitosan/glycerol phosphate hydrogel (CS-GP) was prepared and evaluated as support for enzyme immobilization. In this work a lipase sn-1,3 specific from Rhizopus oryzae was physically immobilized in CS-GP by adsorption (PA) and in CS-GP modified chemically with glutaraldehyde (CS-GP-GA) by covalent attachment (CA). The matrices (pure and chemically modified) and immobilized lipases were characterized by scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and thermogravimetric (TG) analysis. Moreover, a comparative study between free and immobilized lipase was provided in terms of pH, temperature, kinetic parameters and thermal stability. The catalytic properties of biocatalysts were assayed in olive oil hydrolysis. The maximum activities were found at 60◦C/pH 8.5 and 50◦C/pH 7.5 for lipase immobilized by PA and CA, respectively. Both immobilization process turned biocatalysts highly active, since they were stable for 120 days at low (-10◦C) temperatures and around 2-fold more stable than free lipase at 60◦C (high temperature). Thus, results revealed the potential application of chitosan/glycerol phosphate hydrogel (CS-GP) as matrix for lipase immobilization. Nevertheless, better performance was achieved when the lipase was immobilized by covalent attachment.