Human Papillomavirus (HPV) is the principal cause of cervical cancer, which is still a prime universal health concern, especially in underdeveloped nations where access to advanced screening techniques is often restricted. Herein, we propose an electrochemical immunosensor for the determination of HPV 16 L1 protein, a key biomarker associated with cervical carcinogenesis. The developed immunosensor, employing glassy carbon electrode (GCE) enhanced with gold nanoparticles, provides a highly sensitive, specific, and cost-effective approach for early screening of cervical cancer. An optimized stepwise fabrication process which includes electrode surface modification and antibody immobilization, was used to enhance sensor performance. Characterization studies confirmed the effective modification of electrodes and the selectivity of immunoreactions. With an impressive limit of detection (LOD) of 0.033 fg mL−1, the immunosensor showed a linear response to L1 protein concentrations ranging from 0.1 fg mL−1 to 1 ng mL−1. Additionally, the immunosensor exhibits excellent specificity, stability, reproducibility, and regeneration capabilities. The performance of the immunosensor was compared with the enzyme-linked immunosorbent assay (ELISA) by analyzing clinical samples (positive: n = 12; healthy: n = 5) showed satisfactory results. The findings indicated that the proposed sensing platform has the potential to be utilized for the early diagnosis of cervical cancer.