Background. Development of a test card based on up-conversion phosphor technology-based immune lateral flow (UPT-LF) assay as a near-patient detection tool for serum Prothrombin induced by vitamin K absence or antagonist II (PIVKA-II). Methods: Up-converted phosphor nanoparticles (UCPs) were used to bind to PIVKA-II monoclonal antibodies as labeled probes to develop the test card for detecting serum PIVKA-II. The UPT-LF test card was evaluated by the limit of detection, linearity, stability, recovery rate, precision and interference. Preliminary clinical validation was conducted by detection of 498 serum samples from 228 patients with hepatocellular carcinoma (HCC), 170 patients with liver benign lesion (LBL) and 100 healthy controls (HC). Additionally, the correlation of serum PIVKA-II detection between UPT-LF assay and Chemiluminescence enzyme immunoassay (CLEIA) assay were performed. Results: Modified and activated UCPs bound to monoclonal antibodies powerfully to form the luminescent labeled probes. Limit of detection and linear range of UPT-LF test card for serum PIVKA-II were 2.66 and 4.8-20,000 ng/ml, respectively. Test card had good 25 degrees C thermal and 4 degrees C validity period stability, 93.1%-99.2% of recovery rate, 2.6-5.8% and 5.4-8.9% of intra-assay and inter-assay CVs, and strong anti interference ability for 8 common serum analytes. The sensitivity and specificity (vs LBL + HC group) of test card for HCC were 71.5% and 88.9%, respectively. The R2 between UPT-LF assay and CLEIA assay was 0.901. Conclusions: UPT-LF assay provides a reliable, rapid and convenient test for quantitative detection of serum PIVKA-II as well as diagnosis of HCC by a point of care testing way.