Chitosan, sodium alginate, sodium alginate/gelatin and sodium alginate/carboxymethyl cellulose were used as immobilization carrier materials to investigate the immobilization methods of β-galactosidase, and the synthesis of galacto-oligosaccharides (GOS) by the suitable immobilized enzyme was studied. The results showed that the obtained suitable immobilization carrier was chitosan, and the optimal immobilization conditions for β-galactosidase were chitosan mass concentration of 0.03g/mL, standing time of 2h, glutaraldehyde volume fraction of 2%, crosslinking time of 3h, adsorption time of 12h, immobilization temperature of 60℃, enzyme dosage of 0.4mg/g (based on the mass of chitosan microspheres). Compared with the free enzymes, the stability of immobilized enzymes against acid, alkali and heat was significantly improved. Chitosan immobilized β-galactosidase was further used to synthesize GOS, and the optimal reaction conditions were determined as follows:amount enzyme of 2U/mL, pH of 6.0, reaction temperature of 50℃, initial lactose mass concentration of 500g/L, and reaction time of 14h. Under these conditions, the GOS yield reached 52.61%, which was higher than those reported GOS yield (25.1%~46.0%). And the content of GOS with polymerization degree greater than or equal to 3 in the product was 37.07%. After repeated use of the immobilized enzyme for five times, the GOS production activity remained 97.21%. The chitosan-immobilized β-galactosidase obtained from the study had a promising application in the field of GOS synthesis.