Deproteinization of natural rubber using an alkaline protease from Bacillus sp. immobilized on cross-linked chitosan beads is reported. Conditions were identified for attaining a high activity of the immobilized enzyme on the beads. The optimally produced enzyme beads were then used to establish the conditions for effective deproteinization of natural rubber. A two-level full factorial design was used to quantify the main effects and the interaction of the factors influencing the formation of the catalytic beads and the rubber deproteinization process. Results showed that by using the optimal deproteinization process, the total nitrogen level in the rubber was reduced from 0.35% to 0.013% w/w, or a >96% nitrogen removal. In contrast, the urea method of deproteinization achieved a lower nitrogen removal of <86%. The immobilized protease could be used for at least five repeated deproteinization cycles without losing more than 10% of the initial enzyme activity.