Hydrogen peroxide (H2O2) is a bioactive molecule that is involved in many biological and physiological processes, thus the detection of H2O2 is necessary for biological systems, foods, and medicals. We developed a novel phosphor-based film of Li+ co-doped CaWO4:Tb3+/PVA (CWTLP) as a sensor for detection of H2O2 in biological samples. The developed sensor exhibits a green emission peak at 545 nm upon excitation at 255 nm, corresponding to Tb3+ (5D0 →7F2) transitions. The addition of poly vinyl alcohol (PVA) improves the fluorescence properties and enhances lifetime of green light due to bonding between Li+ co-doped CaWO4:Tb3+(CWTL) and PVA, as confirmed by Fourier transform infrared spectroscopy (FTIR). Importantly, the luminescence intensity of the green emission of CWTLP/Ag NPs phosphor's is quenched by silver nanoparticle (Ag NPs) and only recovered by H2O2. These findings point to the mechanism of turn-off-on luminescence behavior. A fluorescence probe and colorimetric method are emphasized to facilitate detection of H2O2 (≤ 5 s) with linear range of H2O2 is 50 to 250 µM (R2 = 0.99) and low limit of detection of 12.8 µM. This optical sensor provides the possibility for detection of H2O2 in human blood serum.