The quantum yield (QY) of semiconductor quantum dots (QDs) is severely hampered by the inherent fluorescence intermittency. The QY of QDs typically increases with an increase in the excitation wavelength. Here, we present a distinctive behavior, where the QY is found to decrease with an increase in the excitation wavelength in water-soluble CdTe QDs (CQDs). Single-particle level measurements highlight the increase in permanent single dark particles at longer wavelengths that comprehend the overall QY of the CQDs in bulk solution. Fluorescence correlation spectroscopy further revealed an increase in the number of dark particles at longer wavelengths. As confirmed by D2O/H2O exchange, the presence of H+ ions in water plays an important role in creating variable permanently dark states in the CQDs. This observation was further supported by the cell internalization study of the CQDs, where a much brighter image at a shorter wavelength than at a longer wavelength was observed. A study of the excitation wavelength-dependent QY in QDs may reveal new insights into the applicability of QDs in different device fabrication cases.