Fluorescence- based lateral flow immunoassays (LFIAs) remain relatively unexplored compared to colorimetric LFIAs for point-of-care (PoC) disease diagnosis and health monitoring. For fluorescence-based LFIAs, a major challenge includes the auto-fluorescence of the nitrocellulose and nonspecific binding of fluorescent polystyrene microspheres. In this paper, we aim to characterize antibody-microsphere conjugates in a fluorescence-based serological assay on nitrocellulose. Factors such as coating concentration and quantity of microspheres were considered and their impacts on nonspecific binding and signal-to-noise ratio are discussed. Finally, we use the determined conditions for the antibody-microsphere conjugates to demonstrate the sensitivity of a proof-of-concept assay detecting antibodies to Epstein-Barr Nuclear Antigen-1 in pooled human plasma samples. A titration of the seropositive plasma samples demonstrated a titer approaching 1:1,000 using only 30 μL of diluted sample and a sample-to-result time of less than one hour.