Plasmonic enhanced fluorescence (PEF) technology is a powerful strategy to improve the sensitivity of immunofluorescence microarrays (IFMA), however, current approaches to constructing PEF platforms are either expensive/time-consuming or reliant on specialized instruments. Here, we develop a completely alternative approach relying on a two-step protocol that includes the self-assembly of gold nanoparticles (GNPs) at the water–oil interface and subsequent annealing-assisted regulation of gold nanogap. Our optimized thermal-annealing GNPs (TA-GNP) platform generates adequate hot spots, and thus produces high-density electromagnetic coupling, eventually enabling 240-fold fluorescence enhancement of probed dyes in the near-infrared region. For clinical detection of human samples, TA-GNP provides super-high sensitivity and low detection limits for both hepatitis B surface antigen and SARS-CoV-2 binding antibody, coupled with a much-improved detection dynamic range up to six orders of magnitude. With fast detection, high sensitivity, and low detection limit, TA-GNP could not only substantially improve the outcomes of IFMA-based precision medicine but also find applications in fields of proteomic research and clinical pathology.