Human Papillomavirus (HPV) is an often asymptomatic widespread sexually transmitted infection responsible for causing various health issues. Low-risk HPV primarily causes genital warts. High-risk HPV types are associated with several cancers, including cervical, anal, and oropharyngeal cancers, posing significant health risks. In this work, we developed an electrochemical biosensor for the detection and differentiation of HPV genotypes based on electropolymerized polypyrrole (PPy) and PAMAM dendrimer-coated gold nanoparticles (PAMAM-AuNPs) for the immobilization of a DNA probe for detecting different HPV genotypes. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and atomic force microscopy (AFM) were used to characterize the biosensor. AFM analysis revealed varied topographic surfaces associated with the increased peaks concerning the biosensor and against patient samples. Electrochemical responses indicated that the genosensor could detect HPV using plasmid (HPV 6, 16, 18, 31, and 33) and cDNA samples (HPV 6, 18, and 31) from infected patients. Different electrochemical profiles were obtained between high-risk and low-risk genotypes. The sensor presented an excellent analytical performance, presenting a lower LOD of 0.04 pg.µL−1 and 0.34 pg.µL−1 for plasmidial and cDNA samples, respectively. Electrochemical analysis pointed out the ability of the developed genosensor platform to differentiate the HPV genotypes. The proposed biosensor is a promising tool for detecting and monitoring HPV and related diseases such as cervical cancer.