Human papillomavirus type 16 (HPV-16) is a key driver in the development of cervical carcinoma, with the integration of its genome into the host DNA marking a critical step in disease progression. Monitoring the physical state of HPV-16, particularly the transition from episomal to integrated forms, is essential for evaluating the risk of malignancy development in cervix. This study presents the development of a duplex electrochemical biosensor for the simultaneous detection of the E2 and E6 genes of HPV-16. Using a one-step sandwich hybridization assay, the biosensor was able to detect HPV-16 E2 and E6 genes with a sensitivity of 8 copies/mL and 12 copies/mL respectively and distinguish between the episomal and integrated forms based on the E2/E6 ratio (cut-off 0.77, 100% sensitivity/specificity). The sensor was validated with 30 clinical cervical tissue samples, providing results comparable to qPCR method. This novel biosensor offers a rapid and efficient platform for the detection and monitoring of HPV-16, with potential applications in cervical cancer screening and prognosis.