Detection of high-risk human papillomavirus (HR-HPV) is critical for early warning and accurate screening of cervical disease in women. Electrochemical biosensors offer a promising analytical strategy that can circumvent the structural complexity of traditional optical components found in current bioassays. In this work, an innovative electrochemical biosensor for HPV-16 detection was developed, utilizing advanced nanomaterial technology and optimized DNA probe grafting processes. The self-assembled composite nanofilm was optimized by conducting an orthogonal analysis. The nanofilm exploited the unique advantages and outstanding performance of (3-aminopropyl)triethoxysilane (APTES) and carbon quantum dots (CQDs), which act as a “molecular bridge” between the biosensing interface and the electrode substrate. The electroactive molecule [Ru(NH3)6]3+ facilitated the highly sensitive signaling of the target DNA by interacting with the double-stranded DNA. Under optimal conditions, the developed electrochemical DNA biosensor exhibited high sensitivity in detecting HPV-16, with a limit of detection (LOD) of 0.73 fM, which showed exceptional potential to be used in widespread point-of-care (PoC) screening programs, opening up new opportunities for various applications.