Point-of-Care (PoC) diagnostic devices, such as lateral flow tests, are often used in low and middle-income countries (LMIC) for low-cost disease detection. Most commercial lateral flow tests use colorimetric detection on a nitrocellulose substrate. In this paper, we present a multistep, fluorescence-based assay detection system, which can detect antibodies in plasma to recombinant protein. Fluorescence-based detection allows us to achieve higher sensitivity, while a nitrocellulose substrate enables fluid handling, high protein immobilization, rapid detection time, and affordability. As a proof-of-concept for detection of disease-specific biomarkers in plasma, we demonstrate the detection of antibodies in plasma to Epstein-Barr nuclear antigen-1 (EBNA-1) recombinant protein and to human papillomavirus (HPV) 16 E7 recombinant protein. We show that our detection system is able to detect EBNA-1-specific antibodies at a 1:10,000 plasma dilution and HPV 16 E7-specific antibodies at a 1:5,000 plasma dilution, indicating high sensitivity. This platform is a low-cost device that can detect fluorescence from labeled biomarkers on a lateral flow assay. Ultimately, we aim to adapt this system to detect HPV 16 and 18 biomarkers for cervical cancer screening in LMICs.