Display omitted •We developed a biosensor platform based on an antigenic colloidal suspension as a biorecognition element.•The platform detects anti-cardiolipin (anti-C) antibodies in infected human serum.•The biosensor was able to detect the formation of immunocomplexes in serum samples.•We obtained a limit of detection of 1024 titer. Syphilis is a sexually transmitted disease with high morbidity and mortality rates. Venereal disease research laboratory (VDRL) and rapid plasma reagin (RPR) testing are the most commonly used syphilis identification methods; however, both have low specificity. The available molecular assays are expensive and time-consuming. Electrochemical biosensors are an innovative approach for detecting multiple target analytes present in syphilis complex samples. In this work, we used a self-assembled monolayer (3-mercaptopropyl)trimethoxysilane (MPTS) to immobilize an antigenic colloidal suspension (containing cardiolipin, cholesterol, and lecithin) as a biorecognition element in a platform for detecting anti-cardiolipin (anti-C) antibodies in infected human serum. The biosensor platform was evaluated through cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. Microscopic evaluation revealed the formation of small cardiolipin-anti-C complexes at the maximum titer of 1:32, thus indicating the serum’s syphilis infection positivity. Anti-C antibody detection was evaluated by blocking electron current and oxidation–reduction processes at the electrode–electrolyte interface. The proposed sensor yielded a linear response (serum dilutions ranging from 1:8 to 1:1024 titer) with a regression coefficient of 0.97. We obtained a limit of detection of 1:1024 titer and high selectivity against interfering biomolecules. The developed biosensor may provide a promising alternative for syphilis diagnosis and follow-up treatment.