In this study, we have developed a simple, cost-effective, label-free fluorescence analytical assay - comprising an adenosine-binding aptamer (Apt(Ado)), platelet-derived growth factor (PDGF)-binding aptamer (Apt(PDGF)), gold nanoparticles (Au NPs), and the DNA-binding dye Oligreen (OG) - for the determination of adenosine. Apt(Ado) and Apt(PDGF) are for the recognition of adenosine and for the amplification of fluorescence signal, respectively. The presence of adenosine induces the conformational switch of the Apt(Ado) from coiled to a G-quadruplex structure, leading to the less binding of Apt(Ado) onto the surface of Au NPs. The more the adenosine is present, the less the amount of Apt(Ado) is adsorbed, resulting in the fluorescence change of the aptamer-OG complexes. When using a mixture of Apt(Ado) (15.0 nM), Au NPs (0.1 nM), and OG (0.05 x) in 5.0mM phosphate (pH 7.4), this sensor provides the limit of detection of 70.0 nM for adenosine at a signal-to-noise ratio of 3. The LOD for adenosine is down to 5.5 nM when using Apt(Ado) modified Au NPs (Apt(Ado)-Au NPs) and Apt(PDGF) for the enrichment of adenosine and amplification of fluorescence signal of OG, respectively. The practicality of the present sensor has been validated by the determination of adenosine in diluted urine samples, showing its advantages of simplicity, selectivity, sensitivity, and minimal matrix interference.