In the present work, spongy graphitic carbon nitride (GCN) and silver oxide nanocomposites were prepared through a facile hydrothermal method at 160 °C for 4 h using GCN, silver nitrate, and dipotassium hydrogen phosphate as the starting materials. The prepared samples were characterized by scanning electron microscopy, energy dispersive X-ray spectrometry, Brunauer-Emmett-Teller method, X-ray diffraction, X-ray photoelectron spectroscopy (XPS), UV-Visible diffuse reflectance spectroscopy, Fourier transform infrared spectroscopy, Raman, and photoluminescence techniques. SEM images showed AgO loaded GCN nanocomposite has a sponge-like structure due to the interconnecting of the enormous layer on the planar structure of GCN. XRD of samples showed the diffraction planes due to the hexagonal structure of carbon nitride with a decrease in intensity of peaks with increasing silver oxide (AgO) in the nanocomposite. Further, the addition of silver oxide improved the electrical properties of the nanocomposite by reducing the recombination of electron and hole pairs as shown by photoluminescence spectra. XPS spectra have confirmed the oxidation state of Ag as well as the coexistence of AgO and GCN in the nanocomposite. BET results indicated the increase in surface area for AgO/GCN-4.2% nanocomposite as compared to GCN. FTIR study indicated that the graphitic structure in GCN remained intact with the loading of AgO. A fluorescent quenching based HO sensor was constructed by simultaneous oxidation of Rhodamine B in the presence of hydrogen peroxide and nanocomposite as the catalyst. In phosphate buffer saline at room temperature, Rhodamine B displayed a strong fluorescence emission peak around 577 nm under an excitation wavelength of 554 nm. This fluorescence signal of Rhodamine B was quenched in the presence of HO and nanocomposite. The catalytic fluorescence quenching was increased with the increase in HO concentration in the reaction system. The detection conditions of the prepared sensor were optimized as a reaction temperature of 25 °C, Rhodamine B concentration as 66 ng mL and nanocomposite concentration as 56 µg mL. The catalytic fluorescence quenching response of the biosensor exhibited a linear range and limit of detection for HO as 30-300 nM and 22 nM respectively.