Abstract Ribonuclease H (RNase H) is an endonuclease which has potential value in the study of anti-HIV drugs. Herein, we report an RNase H assay biosensor by employing the RNA mimics of Green Fluorescent Protein (GFP) like fluorophore for the first time. A hairpin nucleic acid strand (HNS) which modified with phosphorothioate-based antisense oligonucleotides (ASO) to the RNA sequences at the stem part and at the loop part, and a well-designed RNA probe which contains 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) aptamer (denoted “Spinach”) were employed. In the present of RNase H, the HNS RNA sequences and the ASO sequences released in an active form. Then the ASO sequences recognize the probe loop part to form ASO/probe duplexes and open the probe rigid states. After also cleaved by RNase H, the RNA part of the probe were digested in to dNTPs. The digestion makes the probes in a “free” states and the probes accept the DFHBI molecules efficiently. The complex will generate fluorescence intensity at the exciting light and the fluorescence intensity reflects the activity of the RNase H. This strategy can be used for the RNase H activity assay with an ultrasensitive in vitro and imaging RNase H activity in single-cell.