In this paper, glutathione (GSH)-coated Zn-doped CdTe quantum dots (QDs) with different particle sizes were synthesized using the “reflow method”, and the interaction mechanism between the two QDs and lactoferrin (LF) was investigated systemically with different spectroscopic methods. The steady-state fluorescence spectra showed that the LF formed a tight complex with the two QDs through static bursting and that the electrostatic force was the main driving force between the two LF–QDs systems. The complex generation process was found to be spontaneous (ΔG < 0) and accompanied by exothermic and increasing degrees of freedom (ΔH < 0, ΔS > 0) by using the temperature-dependent fluorescence spectroscopy. The critical transfer distance (R0) and donor–acceptor distance (r) of the two LF–QDs systems were obtained based on the fluorescence resonance energy transfer theory. In addition, it was observed that the QDs changed the secondary and tertiary structures of LF, leading to an increase in the hydrophobicity of LF. Further, the nano-effect of orange QDs on LF is much larger than that of green QDs. The above results provide a basis for metal-doped QDs with LF in safe nano-bio applications.