In this work, a new platform for effective sensing cysteine (Cys) was developed based on fluorescence resonance energy transfer (FRET) between FAM-tagged single-stranded DNA (FAM-ssDNA) and graphene oxide (GO). Due to the noncovalent assembly between FAM-ssDNA and GO, fluorescence quenching of the FAM took place because of FRET. This method relied on the competitive ligation of Ag+ by Cys and “cytosine–cytosine” (C–C) mismatches in a FAM-labeled DNA strand of the self-hybridizing strand. At first, enough amount of Ag+ was introduced to bind “C–C” mismatches and form double-stranded DNA (dsDNA), which had weak affinity to GO and kept FAM away from GO surface. However, the presence of Cys removed Ag+ away from “cytosine–Ag+–cytosine” (C–Ag+–C) base pairs, leading to the formation of ssDNA again and FRET, and then fluorescence of the FAM-ssDNA was efficiently quenched. The fluorescence intensity decrease was found to be proportional to the increase of concentration of Cys in both aqueous buffer (2–200 nM) and human serum (5–200 nM), and the sensitivity of the proposed method towards Cys was much higher than that of other reported assays for Cys.Highlights► A new platform for fluorescent detecting cysteine was designed. ► FRET between FAM-ssDNA and GO was researched to construct the method. ► Detection of cysteine has been effectively realized in buffer and serum.