In this work, we have developed a simple and highly sensitive fluorescent biosensor for the detection of HIV-1 RNase H activity and inhibition by using graphene oxide (GO) as sensing platform. In our approach, a DNA-RNA substrate was prepared which a carboxy fluorescein (FAM) was labeled at the 3 termini of single strand DNA. The FAM labeled DNA-RNA substrate preserves most of the fluorescence when mixed with GO. However, in the present of RNase H, RNase H can cleavage the RNA into fragments, resulting in the dissociation of ssDNA and mononucleotides. The fluorescence intensity was greatly quenched after the addition of GO. Because of the super quenching ability of GO, the as-proposed method provides a wide linear range from 1.0 to 26.3 units mL–1 and a low detection limit of 0.6 units mL–1 for HIV-1 RNase H activity analysis. Additionally, this approach shows potential for high-throughput screening of HIV-1 RNase H inhibitors. The method not only provides a universal platform for monitoring activity and inhibition of RNase H but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.