The development of simple, sensitive and cost-effective methods for specific nucleic acid detection has attracted tremendous attention due to its importance to the early diagnosis of genetic diseases and to biodefense applications. In this work, we demonstrated a fluorescent turn-off mode DNA assay based on l-cysteine-modulated synthesis of CdTe quantum dots (CdTe QDs), horseradish peroxidase-mimicking G-quadruplex-hemin-K complex controlled oxidation of l-cysteine to cystine, and catalyzed hairpin assembly (CHA)-assisted signal amplification. After the addition of target DNA, the CHA signal amplification reaction was triggered and numerous H1-H2 double-stranded DNA were formed, initiating the release of G-quadruplex sequences in H2 simultaneously. Thus, the degree of inhibition of the synthesis of CdTe QDs is proportional to the concentration of the G-quadruplex sequence in this method. In contrast, when the target DNA was absent, the CHA could not be triggered, and the fluorescence signal was high due to the remaining intact l-cysteine. Under optimal experimental conditions, the homogeneous fluorescence method achieved the detection of HIV DNA with a linear range from 0.1 pM to 1 nM and a detection limit of 0.12 pM. This novel biosensor exhibits excellent specificity in differentiating DNA sequences with a single-base and two-base mismatch. To the best of our knowledge, this a label-free and highly sensitive bioassay utilizing CHA-assisted signal amplification and G-quadruplex control of synthesis of CdTe QDs strategy was not reported in previous. Thus, this proposed strategy is anticipated to find use in basic biochemical research and clinical diagnosis.