A novel bio-electrochemical immunosensor for sensitive and selective detection of anti-peptide antibodies of HPV was fabricated by immobilization of a peptide (SPINNTKPHEAR) (1) linked to a 6-aminohexanoic (Ahx) residue and ferrocene (Fc) on a gold electrode surface, used as sensing interface. The Fc–Ahx–peptide (Fc–Ahx–SPINNTKPHEAR) (1AFc) was obtained by solid phase peptide synthesis by using the Fmoc/tBu strategy. Peptides were purified by preparative HPLC, characterized by MS, MALDI-TOF, and Circular Dichroism and their purity was evaluated by using analytical HPLC. The electrochemical behavior of the modified electrode was examined by cyclic voltammetry in phosphate buffer solution (PBS) in order to evaluate the redox behavior of the ferrocene moiety. The influence of anti-peptide antibodies on the voltammetric response of the modified electrode was investigated by comparing results obtained with pre-immune (control) and post immune serum samples at similar dilution factor. Changes in such behavior upon addition of the serum samples to PBS suggested that the fabricated bioelectrochemical sensor was able to recognize the interaction between anti-peptide antibodies and the immobilized peptide (1AFc) with high selectivity and sensitivity. Such influence increased as the dilution decreased, but the effect was less pronounced at relatively high concentrated solutions owing to the effect of the sample matrix. Notwithstanding, the proposed biosensor can clearly detect the target anti-peptide antibodies at a significant large concentration range.