Tumor molecular subtyping is essential for guiding personalized treatment strategies. However, existing detection methods are constrained by complex procedures, prolonged processing times, and high costs. Therefore, the development of rapid and straightforward detection methods remains a critical unmet need. Here, we employed DNA nanotechnology to construct a series of fluorophore-coupled pairs (a chiral BODIPY (cBDP) and a cyanine), revealing intermolecular interactions via various spectroscopic measurements and identifying a pair with unique charge and chirality transfer properties. By the integration of the fluorophore pair as a probe into DNA circuit systems, it can achieve amplification of dual optical signals of circular dichroism (CD) and fluorescence through DNA cascade reaction-controlled dissociation and the formation of the probe. For the practical implementation, we applied this dual-signal detection system for breast cancer molecular subtyping, which proved to successfully detect key biomarkers, estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2), within 1 h. The receptor-specific spectral responses enabled the rapid classification of four breast cancer molecular subtypes. Particularly, the synergistic effect of charge and chirality transfer proved to contribute to the enhancement of the detection accuracy. Given its versatility and precision, this platform shows significant promise for tumor molecular subtyping and offers potential applications in clinical biomarker detection and personalized therapy.