Human papillomavirus type 16 (HPV16) is a primary etiological agent of cervical cancer, underscoring the importance of early detection, especially of E7 mRNA expression, for effective clinical management. In this study, we developed a fluorescence-based nanobiosensor for the ultrasensitive and specific detection of HPV16 E7 mRNA. The catalytic hairpin assembly (CHA) was integrated with Fe 3O 4@Au core-shell nanoparticles (Fe 3O 4@Au NPs), enabling the combined advantages of magnetic enrichment and enzyme-free signal amplification. The nanobiosensor exhibited a linear fluorescence response over the ranges of 0.002 to 1 pM in PBS buffer and 0.1 to 1 pM in first-void urine (FVU) following magnetic separation. This approach demonstrated excellent reproducibility, high specificity with the ability to discriminate single-base mismatches, and stability for up to 45 days. The performance of the nanobiosensor was successfully validated in RNA extracted from HPV16 plasmid-transformed E. coli, CasKi cells, and clinical swab specimens. Validation of the developed nanobiosensor was conducted through the analysis of clinical samples, demonstrating complete concordance with results obtained from commercial diagnostic assays. These results highlight the potential of this nanobiosensor as a robust tool for early detection and monitoring of HPV16 infections.