Anti-human papillomavirus (HPV) antibody detection is promising technique for detecting women at risk of suffering cervical cancer, since potentially oncogenic, persistent, long-term HPV-infections elicit an antibody response which is rarely detected in transitory HPV-infection patients. We have identified a non-variable C-terminus L1-peptide, belonging to an α-helix surface exposed on L1-protein, specifically recognized by antibodies from HPV-associated cervical lesion patients. This peptide tested against 313 sera presented higher reactivity with antibodies from cervical cancer (OD mean 0.43 ± 0.13) or cervical lesion patients (OD mean 0.41 ± 0.17) than antibodies from normal cytology patients (OD mean 0.17 ± 0.03). High-risk HPV-infected patients presented higher antibody reactivity (OD mean 0.36 ± 0.17) than high-risk HPV-non-infected patients (OD mean 0.22 ± 0.11). This peptide showed 88.36% sensitivity, 99.39% specificity and 94.21% correct classification of high risk-HPV cervical lesion or cervical cancer patients. This peptide should be taken into account for designing serological screening or diagnostic tests for use in a clinical scenario.