G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. There is considerable interest in the design of ligands that target G-quadruplex DNA because of their potential anticancer activity. We designed a fluorescence resonance energy transfer (FRET) system to identify molecules that stabilize G-quadruplexes in a homogeneous medium using unmodified gold nanoparticles (GNPs) as a fluorescence quencher. The assay exploits the different adsorption abilities of GNPs for single-stranded DNA and double-stranded DNA. Fluorescein-tagged probe DNA adsorbed onto the surface of GNPs can quench the fluorescence of a DNA probe. Intramolecular folding of an oligonucleotide of the human telomeric sequence into a G-quadruplex structure led to fluorescence enhancement in the presence of quadruplex-binding ligands. G-quadruplex formation, induced by specific binding of GDNA ligands, was investigated by CD measurements. Melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the absorbance at 295 nm. Two series of natural drugs were studied, and flavonoids were shown to increase the melting temperature of the G-quadruplex. This increase in the Tm value was well-correlated with an increase in FRET efficiency. The combined data from fluorescence measurements and melting experiments indicate that the FRET approach offers a simple, sensitive, and effective method to identify ligands with potential anticancer activity.