The RNase H activity of HIV-1 reverse transcriptase was examined using chemically synthesized deoxyribo ribo-oligonucleotide hybrid duplex labeled with the fluorescence donor at the 5′-end and with the fluorescence acceptor at the 3′-end of DNA strand as a substrate. Fluorescence resonance energy transfer (FRET) between these fluorescent dyes was used to analyze the rate of the enzymatic reaction. Under excitation of the donor dye, that is 6-carboxyfluorescein (6-FAM), at 490 nm, the increase of the fluorescence resulting from the acceptor dye, that is 6-carboxytetramethylrhodamine (TAMRA), at 578 nm, was observed depending on the degradation of DNA-RNA hybrid duplex. This method can be introduced into the high throughput screening of the inhibitors against the RNase H activity for anti-HIV drug.