NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome is a multiprotein complex that plays a crucial role in host immune responses to both pathogen infections and sterile injuries. Construction of the NLRP3 inflammasome system in HEK293T cells provides a more convenient and concise method for studying the regulation mechanisms of inflammasome activation. Plasmids NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, and pro-1β were transfected into HEK293T cells. Subsequently, the mature IL-1β levels were determined by Western blot and enzyme-linked immunosorbent assay (ELISA) after adding nigericin. Our results showed that the mature IL-1β could be detected in cell lysates and supernatants of cells transfected with intact NLRP3 inflammasome components whereas not in cells transfected with incomplete NLRP3 inflammasome components. Meanwhile, the mature IL-1β increased with the elevated amounts of transfected NLRP3. In this study, NLRP3 inflammasome system was successfully constructed in HEK293T cells, which laid a foundation for future research.