We developed a simple, sensitive inner filter effect (IFE)-based fluorescent assay for sensing H2O2 and cholesterol. In the process, poly(vinylpyrrolidone)-protected gold nanoparticles (PVP-AuNPs) and fluorescent BSA-protected gold nanoclusters (BSA-AuNCs) were used as an IFE absorber/fluorophore pair. PVP-AuNPs can be a powerful absorber to influence the emission of the fluorophore, BSA-AuNCs, in the IFE-based fluorescent assays. That is due to the high extinction coefficient of AuNPs and the complementary overlap between the surface plasmon resonance (SPR) absorption of PVP-AuNPs and the excitation of BSA-AuNCs. The PVP-Au seeds, produced by directly mixing PVP with HAuCl4, were able to catalyze H2O2 to enlarge AuNPs. The SPR absorption of PVP-AuNPs was enhanced with an increased concentration of H2O2 and, subsequently, induced significant fluorescence quenching of BSA-AuNCs. The IFE-based fluorescent assay enabled the detection of H2O2 and generation of H2O2 in the presence of O2/cholesterol and cholesterol oxidase (ChOx) by the fluorescence response of BSA-AuNCs. The present IFE-based approach can detect H2O2 ranging from 1 to 100 μM with a detection limit of 0.8 μM and cholesterol ranging from 1 to 100 μM with a detection limit of 1.4 μM.