Novel, water-soluble Zn²⁺ doped CdTe quantum dots (QDs) were prepared through a one-step hydrothermal route. Due to high temperature and high pressure in the hydrothermal route, the addition of Zn ions and employment of a new stabilizer N-acetyl-L-cysteine (NAC), the as-prepared CdTe: Zn²⁺ QDs (emission wavelength at 530–623 nm) exhibit a high quantum yield (up to 75.31%), high doping ratio (the actual constituent ratio reaches 2.41 : 1.00), high stability and excellent biocompitability. The characterization of as-prepared QDs was carried out through fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. In particular, for the first time, we realized qualitative, semi-quantitative and quantitative studies on the doping of Zn to CdTe QDs through X-ray photoelectron spectroscopy, electron diffraction spectroscopy and atomic absorption spectrometry. In addition, the relation between the feed ratio of Zn to Cd and the actual constituent ratio of the prepared QDs was also examined.