G-Quadruplexes formed by G-rich DNA are of broad interest due to their involvement in telomere function, gene transcription and recombination. Small ligands that interact strongly with G-quadruplexes have been considered to further influence telomeric function and gene transcription. Because most G-rich sequences are trapped in duplex structures in gene promoters, ligands that can stabilize G-quadruplexes in the presence of their complimentary strands would likely have strong effects on gene transcription. Here, we report a novel simple small molecule (pyridyl carboxamide), consisting of three pyridine rings and four amide bonds. Comparing with some reported G-quadruplex ligands, this molecule not only selectively stabilizes G-quadruplexes rather than duplexes, but also maintains a G-quadruplex structure even if the G-rich region was trapped in long double-stranded DNA (dsDNA). It is widely believed that the dissociation of duplexes is involved in gene transcription and that the formation of the G-quadruplex influences some oncogene expression. Py-Am exhibited strong G-quadruplex-forming ability within a long dsDNA sequence, suggesting it would have potent effects on the G-quadruplex-forming sequences involved in gene transcription.