Development of a simple electrochemical immunosensor, for the direct detection of biotin, is important for monitoring biotin content. A displacement assay was used in biotin detection, in which surface-bound antibodies were dissociated from the surface of immunosensor in the presence of free biotin. Pre-treatment of samples is not required for biotin detection using this electrochemical immunosensor. Using this electrochemical immunosensor, the recoveries of biotin in the two infant formulas A and B were 93.9% and 91.3%, respectively, of their biotin concentrations as stated on their packaging. The direct detection of biotin, with this electrochemical immunosensor, in supplements X, Y, and Z, showed recoveries as high as 92.9%, 106.5%, and 100.0%, respectively. The accuracy of our electrochemical immunosensor was validated with high pressure liquid chromatography (HPLC). The surface of immunosensor had a strong anti-fouling property and high specificity for actual applications in complex matrices. Additionally, the developed immunosensor shows good stability, reproducibility, and intra- and inter-day precision. This electrochemical immunosensor can directly detect biotin in infant formulas, biotin-containing supplements, and serum.