Graphene quantum dots (GQDs) exhibit excellent optical properties, low toxicity, good biocompatibility, and high re-active oxygen species (ROS) generation efficiency under photoexcitation, demonstrating significant potential for achieving visualized therapy. However, conventional synthesis methods yield GQDs as multi-component mixtures with compromised safety and consistency, coupled with unclear luminescence mechanisms and difficulties in precise microstructure control, which severely hinder their development as nanomedicines. Therefore, it is urgent to synthesize mono-component single-molecule GQDs and systematically investigate the structure-property relationships governing their optical characteristics and ROS generation capabilities. This work established a modular organic synthetic strategy based on electron donor-acceptor (D-A) architectures, successfully preparing three single-molecule GQDs with identical core structures but distinct edge functionalization: 12Me-GQD (12D-A), 12Br-GQD (D-12A), and 6Br-6Me-GQD (6D-π-6A). Systematic characterization reveals that electron-donating groups (-CH3) induce a blue shift in photoluminescence (PL) emission, enhance fluorescence quantum yield, and improved photo-induced ROS production. Conversely, electron-withdrawing groups (-Br) cause emission red shift, reduce PL quantum yield, and completely suppress photodynamic activity. Notably, the alternately substituted 6Br-6Me-GQD exhibits enriched electron transfer pathways, demonstrating dual emission peaks and optimal photodynamic performance. Importantly, both 12D-A type and 6D-π-6A type GQDs maintain effective ROS generation under hypoxic conditions, addressing the critical limitation of conventional photosensitizers in oxygen-deprived tumor microenvironments. This work established a modular synthetic system for single-molecule GQDs with different D-A types and revealed the structure-property relationships governing optical characteristics and ROS generation capabilities, providing novel insights for the development of hypoxia-adaptive PDT nanomedicine.