<p>Upconversion nanoparticles (UCNPs)-based fluorescence lateral flow immunoassay (FLFIA) has significant application prospects in clinical diagnosis, but it still suffers from relatively low sensitivity in detecting trace amounts of biomarkers. Here, we report for the first time a strong fluorescence-emitting core-shell-shell NaLuF₄@NaLuF₄:Yb,Er@NaLuF₄ UCNP as the FLFIA probe. The UCNPs-based FLFIA platform enables point-of-care detection of cardiac troponin I (cTnI) with high sensitivity, accuracy, and resistance to autofluorescence interference. NaLuF₄:Yb,Er encapsulated in the intermediate shell layer can effectively reduce the cross-relaxation and surface quenching of the luminescence center, resulting in a higher quantum yield. Compared with the conventional core-shell NaLuF₄:Yb,Er@NaLuF₄ UCNPs, the fluorescence intensity increases by 2.4 times. The UCNPs-based FLFIA was further integrated with a portable smartphone platform for rapid, on-site detection. The UCNPs probe enabled sensitive cTnI detection, with a detection limit of 0.035&#xa0;ng/mL in buffer and 0.059&#xa0;ng/mL in serum, and a detection range of 0.05–100&#xa0;ng/mL. In testing clinical samples, the results from the platform showed excellent correlation with hospital results. We anticipate that the NaLuF₄@NaLuF₄:Yb,Er@NaLuF₄ UCNPs can serve as a promising immunolabeling nanoprobe for highly sensitive and accurate FLFIA detection.</p> Graphical Abstract <p></p>