Procerain B is a novel cysteine protease isolated from Calotropis procera by our group and published recently. We have further characterized the enzyme by N-terminal sequencing and peptide mass fingerprinting. Procerain B showed maximum sequence similarity (80%) with Asclepain. Moreover, the characteristic VDWR motif of cysteine proteases is present in procerain B. The N-terminal and peptide mass fingerprinting analysis showed a distinct nature of the enzyme. Various applications of the enzyme were also evaluated. Procerain B is very effective in milk-clotting and may be a potential candidate for this process in the cheese industry. Additionally, the enzyme has potential application as dietary supplement to aid digestion. Effects of various metal ions on milk-clotting activity were also studied. The milk-clotting activity was increased in case of few metals while others have a negative effect. It is worth mentioning that the easy availability of plant material and simple purification method makes industrial production of the enzyme feasible. A protease with easy purification and suitable properties for application is always desired.