© 2020 Elsevier B.V.G-quadruplex secondary structures have gained significant recognition due to the discovery of their involvement in regulation of gene expression and their association with many diseases such as cancer and neurological disorders. Consequently, the need for the recognition and characterization of G-quadruplex structures has increased considerably. Here, we present a rapid, facile and sensitive off-on-off in vitro platform for G-quadruplex detection, based on the gold nanoparticle-azacyanine5 (AuNP-Aza5) conjugated fluorescence probe. The conjugated probe is governed by Fluorescence Resonance Energy Transfer (FRET) mechanism between the fluorophore molecule, Aza5, and AuNPs. The fluorescence of Aza5 that was hindered by AuNPs (off), was restored in the presence of L-cysteine (on) until the addition of a G-quadruplex structure (off). The developed sensing platform selectively responds to G-quadruplex structures formed within the promoter regions of VEGF-Pu22, K-RAS, C-myc and BCL-2. It doesn't exhibit a similar response to the other secondary structures such as single, double or triple stranded nucleic acid structures. The detailed investigation of the probe with VEGF-Pu22 as a model G-quadruplex structure revealed a linear response between the concentration range of 0.032–0.347 μM with a detection limit of 12.66 nM.