The early protein HPV E6 of human papillomavirus is the major oncoprotein in high-risk subtypes. Due to its critical role in malignant tumorigenesis, it is a widely recognized biomarker for the early diagnosis of HPV-related cancers. The lack of effective and commercially available monoclonal antibodies (mAbs) against HPV16 E6 makes the sensitive detection of HPV16 E6 a great challenge. In the present study, DNA-protected silver nanoclusters (AgNCs–dsDNA) were used as a sensitive fluorescent probe for the detection of HPV E6. Both the emission enhancement and the blue shift induced by HPV E6 provide a new approach for the detection of this protein in vitro. This method exhibits a high sensitivity (LOD of 0.886 nM), good selectivity and strong anti-interference to several common proteins, which can also be applied for the detection of HPV 16 E6 in bovine serum. Therefore, it shows potential utility in the clinical diagnosis of related cancers. Particularly, the intrinsic mechanism of the highly sensitive response of AgNCs–dsDNA to HPV E6 was explored in detail by choosing several representative peptides from E6, and it was revealed that in addition to the electrostatic interaction between them, the involved cysteine was essential for the specific recognition of this protein; therefore, the response of AgNCs–dsDNA to the protein was due to the synergistic effect of different factors, and the cysteine residue was the dominant factor. The oppositely charged surfaces of E6 and AgNCs–dsDNA attract each other via electrostatic interactions, and then, E6 is anchored onto the surface of the nanocluster via the newly formed Ag–S bond; the present study not only enriches the biological application of metal nanoclusters but also shows significant potential for the early diagnosis of the HPV-induced cancers.