Prostate specific antigen (PSA) is a widely-used biomarker for the diagnosis, screening, and prognosis of prostate cancer (PCa). It is critical to develop a rapid and convenient method to accurately detect PSA levels, especially when the PSA levels are in the clinical gray area of 4-10 ng/mL. We developed a novel upconversion nanoparticle (UCNP)-based fluorescence lateral flow test strip for qualitatively and quantitatively detecting PSA. The carboxyl group-modified UCNPs (UCNP-COOH) were labeled with anti-PSA antibodies via 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as labeling probes to recognize PSA. The fluorescence intensity of the UCNP-probe was then measured with a laser fluorescence scanner. A total of 1397 serum and 20 fingertip blood samples were collected to validate the UCNP strip. A reliable correlation between the area ratio (TC), reflecting the fluorescence intensity of the test/control line, and the PSA concentration was observed (r = 0.9986). The dose-dependent luminescence enhancement showed good linearity in the PSA concentration range from 0.1 to 100.0 ng/mL with a detection limit of 0.1 ng/mL. Our UCNP POCT strip demonstrated excellent accuracy, anti-interference and stability in the gray zone (4-10 ng/mL) of PSA clinical application and outperformed other PSA test strips. The UCNP strip showed good consistency with the Roche chemiluminescence assay in 1397 serum samples. It also showed good performance for PSA detection using fingertip blood samples. This novel UCNP-based test strip could be a sensitive and reliable POCT assay to detect PSA, facilitating the diagnosis and surveillance of PCa.