A new ratiometric electrochemical aptasensor has been developed for highly sensitive and selective detection of bisphenol A (BPA). The double-stranded DNA (dsDNA), consisting of the BPA aptamer (DNA1) and methylene blue (MB)-labeled complementary DNA (cDNA), was immobilized on a gold nanoparticle (AuNP) modified glassy carbon (GC) electrode. In the presence of BPA, the specific BPA–aptamer interaction drove the release of the MB-labeled cDNA from the electrode surface. As a result, the oxidation peak current of MB (IMB) decreased and that of BPA (IBPA) increased. The peak current ratio (IBPA/IMB) of BPA and MB was linear with the concentration of BPA in the range from 1 to 100 pmol/L with a detection limit of 0.6 pmol/L. The detection limit is much lower than that obtained by most of the reported electrochemical methods. On the other hand, the developed aptasensor possesses good selectivity, reproducibility, and stability, and the related sensing structure is very simple, showing promising practical applications in BPA assays.