A novel “off-on” ratiometric fluorescent aptasensor was established for adenosine detection based on fluorescence resonance energy transfer (FRET) between CdS QDs, DNA QDs as donor and graphene oxide (GO) as acceptor. Amino-riched DNA QDs covalently bonded to the carboxyl group on the edge of the GO, and with the absorption of the TGA-modified CdS QDs with aptamer (CdS QDs-apt) onto the GO surface via the π-π stacking interaction. The fluorescence of both CdS QDs and DNA QDs were efficiently quenched due to FRET (turn off). When adenosine was present, the specific binding of the aptamer to the target preferentially that released the CdS QDs-apt from GO. The process would inhibit the FRET which contribute to the fluorescence of CdS QDs-apt recovery again (turn on), while the fluorescence intensity of DNA QDs only slightly altered and acted as the reference signal. Thus, a novel “off-on“ ratiometric fluorescent aptasensor for adenosine detection was constructed accordingly. There was a good linearity relationship between the ratio of the FL intensity (F595 nm/F464 nm) and the concentration of adenosine in the range of 20.00–180.0 nmol/L with a detection limit of 1.3 nmol/L (S/N = 3, n = 9). Importantly, the feasibility of the developed aptasensor for selective detection of adenosine in serum and urine samples with satisfactory results. The recoveries were observed to be 97.04–100.2 %.