Deubiquitinases (DUBs) are a group of enzymes that regulate myriad biological processes by removing ubiquitin posttranslational modifications. They can either cleave ubiquitin directly from a substrate protein or disassembling a ubiquitin chain. Currently, about 100 human DUBs were identified and they were classified into seven families, and majority of them are cysteine proteases. DUBs exhibit distinct activities and preferences towards a wide variety of complex Ub chain with different linkages and architectures. Understanding how DUB achieves the selectivity towards specific substrates is important to elucidate how Ub system is regulated. This dissertation discusses our efforts to investigate the molecular mechanism of DUB, specifically the proteasome associated DUB UCH37/UCHL5. We have combined biochemical assay, biophysical characterization, mass spectrometry, and cell biology to address how DUB interacts with different ubiquitin chains to achieve substrate specificity. These lead us to uncover a novel mechanism that DUBs may use distinct surfaces for distinct activities. Finally, the discovery of the new mechanism of DUB would also provide new avenues for the DUB inhibitors development for therapeutics.