Cardiac troponin I (cTnI) is a significant biomarker for acute heart attack. Hence, fast, economical, easy and real time monitoring of cardiac troponin I (cTnI) is of great importance in diagnosis and prognosis of heart failure in the healthcare domain. In this work, an immunoassay based on NaYF4:Yb/Ho based photon-upconversion nanoparticle (UCNP) with narrow emission peaks at 540 nm and 655 nm respectively, is synthesized. Then, it is encapsulated with amino functionalized silica using 3-aminopropyltriethoxysilane (APTES) to form APTES@SiO2-NaYF4:Yb/Ho UCNPs. When AuNPs is added to this system, the fluorescence is quenched by the electrostatic interaction with APTES@SiO2-NaYF4:Yb/Ho UCNPs, thereby exhibiting a FRET-based biosensor. When the cTnI antigen is introduced into the developed probe, an antibody-antigen complex is formed on the surface of the UCNPs resulting in fluorescence recovery. The developed sensor shows a linear response towards cTnI in the range from 0.1693 ng mL-1 to 1.9 ng mL-1 with a low limit of detection (LOD) of 5.5 × 10-2 ng mL-1. The probe exhibits adequate selectivity and sensitivity when compared with coexisting cardiac biomarkers, biomolecules and in real human serum samples.