Chitosan, sodium alginate, sodium alginate/gelatin, sodium alginate/carboxymethyl cellulose were used as immobilized carrier materials to investigate the immobilization methods of β-galactosidase, and the synthesis of galacto-oligosaccharide (GOS) by the immobilized enzyme was studied. The results showed that the obtained suitable immobilization carrier was chitosan, and the optimal immobilization conditions were deter mined as follows: chitosan mass concentration 0.03 g/mL, standing time 2 h, glutaraldehyde volume fraction 2%, crosslinking time 3 h, adsorption time 12 h, immobilization temperature 60 ℃, enzyme dosage 0.4 mg/g chitosan microspheres. Compared with the free enzyme, the immobilized enzyme""s stability against acid, alkali and heat was significantly improved. Chitosan immobilized β-galactosidase was further used to synthesize GOS, and the optimal reaction conditions were deter mined: amount of enzyme 2 U/ mL, pH 6.0, 50 ℃, initial lactose mass concentration 500 g/L, and reaction time 14 h. Under these conditions, the GOS yield reached 52.61%, which was higher than those reported. And the content of GOS with DP ≥ 3 in the GOS 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 has a promising application in the field of GOS synthesis.