The Plackett-Burman experimental design was applied for screening of the influence of various factors on the immobilization of β-galactosidase on chitosan beads. The steepest ascent method was required the optimum conditions effectively and efficiently to approach the maximum of immobilized enzyme activity. Experimental results indicated that the Plackett-Burman design showed enzyme concentration, adsorption pH, adsorption temperature, glutaraldehyde concentration and cross-linking pH were the positive effect, but adsorption time and cross-linking temperature were the negative effect. Analysis of the results showed that the adsorption time, adsorption pH and cross-linking pH were the main effective parameters on the immobilization of β-galactosidase. Further, the steepest ascent experiment indicated that the immobilized enzyme activity reached 161.85U/g when the adsorption time, adsorption pH and cross-linking pH was 4h, 7 and 7, respectively. And the results increased about 37.60% than the previous single factor experiment.