The development of an analytical method for determining the properties of quantum dots (QDs) is crucial for improving the optical performance of QD-based displays. Therefore, synchrotron-based X-ray photoelectron spectroscopy (XPS) is designed here to accurately characterize the chemical and structural differences between different QDs. This method enables the determination of the reason for the minimal differences between the optical properties of different QDs depending on the synthesis process, which is difficult to determine using conventional methods alone. Combined with model simulations, the XPS spectra obtained at different photon energies reveal the internal structures and chemical-state distributions of the QDs. In particular, the QD synthesized under optimal conditions demonstrates a relatively lower degree of oxidation of the core and more uniformly stacked ZnSe/ZnS shell layers. The internal structures and chemical-state distributions of QDs are closely related to their optical properties. Finally, the synchrotron-based XPS proposed here can be applied to compare nearly equivalent QDs with slightly different optical properties.