In this work, the reduced metal-organic frameworks (MOFs) that can function as a three-dimensional catalytic nanoreactor for copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction have been fabricated for development of the immunoassay. In our design, MOFs with well-defined porosity are accessible to the substrates of CuAAC, while the substrates can diffuse in the pore channel. Moreover, the high-ordered structural alignments of MOFs are beneficial for integration of copper ions into the three-dimensional lattice. Therefore, upon exposure to reduction, the copper nodes in MOFs are transformed from CuII to CuI in situ to achieve spatiotemporal control, eliminating the primarily dissolution procedure. Furthermore, the reduced MOFs can serve as a powerful catalyst to facilitate the CuAAC click reaction, which can greatly reduce time and improve the reaction efficiency. In addition, the proposed immunoassay shows high analytical performance for hepatitis B virus surface antigen (HBsAg) quantification in the linear range of 0.03-2.0 ng mL-1 with a detection limit down to 11.2 pg mL-1. The new immunoassay has also been challenged in detection of clinical serum sample, and a good consistency can be found with the time-resolved fluorescence immunoassay (TRFIA) in clinics, signifying its promising potential in clinical diagnosis.