Abstract Multiplex and rapid detection of biomarkers in gingival crevicular fluid (GCF) is critical to achieve accurate and timely diagnosis of periodontitis severity and progression. Aiming at providing a point-of-care testing (POCT) platform for clinical diagnosis of periodontitis at chair side, we developed a disk-like lateral flow immunoassay strip (LFIS) using green core-shell upconversion nanoparticles (G-UCNPs) as luminescence probe to simultaneously detect three periodontitis biomarkers in GCF, i.e., matrix metalloproteinases-8 (MMP-8), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). The developed G-UCNPs-LFIS platform achieved highly sensitive detection of the three biomarkers in standard solutions with detection limits of 5.455, 0.054 and 4.439 ng/mL, respectively, and exhibited high detection specificity in spiked GCF. A high and stable recovery rate of the three biomarkers rate was also obtained in spiked artificial saliva. Furthermore, the simultaneous detection of the three biomarkers was successfully performed in clinical GCF samples for the first time. And the results showed a relative high correlation index (0.995 for MMP-8 detection, 0.976 for IL-1β detection, 0.977 for TNF-α detection) compared with those obtained using the clinical detection methods, verifying the diagnosis accuracy of the developed G-UCNPs-LFIS. Additionally, the total time to accomplish the detection only took 30 min once, enabling users to timely monitor their periodontitis conditions. We thus envision the great promise of the developed G-UCNPs-LFIS platform for accurate and timely diagnosis of periodontitis in clinic.