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Nitrogen doping and amino group functionalization through chemical modification lead to strong electron donation. Applying these processes to a large <i>π</i>-conjugated system of graphene quantum dot (GQD)-based materials as electron donors increases the charge transfer efficiency of nitrogen-doped amino acid-functionalized GQDs (amino-N-GQDs), resulting in enhanced two-photon absorption, post-two-photon excitation (TPE) stability, TPE cross-sections, and two-photon luminescence through the radiative pathway when the lifetime decreases and the quantum yield increases. Additionally, it leads to the generation of reactive oxygen species through two-photon photodynamic therapy (PDT). The sorted amino-N-GQDs prepared in this study exhibited excitation-wavelength-independent two-photon luminescence in the near-infrared region through TPE in the near-infrared-II region. The increase in size resulted in size-dependent photochemical and electrochemical efficacy, increased photoluminescence quantum yield, and efficient two-photon PDT. Therefore, the sorted amino-N-GQDs can be applicable as two-photon contrast probes to track and localize analytes in in-depth two-photon imaging executed in a biological environment along with two-photon PDT to eliminate infectious or multidrug-resistant microbes.