3CLpro (3C-like protease) is a key protease in coronaviruses, responsible for cleaving multiple protein precursors during viral replication and assembly. This enzyme features a unique three-dimensional structure, consisting of two β-barrel domains. 3CLpro functions primarily in the cytoplasm of host cells, facilitating viral particle maturation and release by cleaving viral polyproteins. Its activity and expression are regulated by both viral genome and host cell factors. Mutations in 3CLpro can affect its catalytic activity or drug sensitivity, significantly impacting the virus life cycle and drug resistance.