A novel strategy to prepare a selective ultrathin molecularly imprinted polymer (MIP) film directly on the gold-based transducer surface for the peptide and protein detection in aqueous solution is demonstrated using a combination of epitope- and electrochemical surface imprinting approach. The synthetic peptide derived from the surface-exposed C-terminus of cytochrome c (Cyt c, residues 96–104) is selected as the template for the imprinting. It is labeled with a fluorescent dye in order to quantitatively evaluate all stages of the imprinting process in terms of changes in mean fluorescence intensity. The labeled peptide template is first chemisorbed on the gold surface as an oriented submonolayer through an additional C-terminal cysteine. After electropolymerization, the template is stripped off electrochemically. To allow the imprinted sites to be confined to the surface, the film thickness is controlled to be comparable to the thickness of the peptide layer. This is achieved by the electropolymerization of scopoletin. Recognition capabilities of the films are characterized and the resulting MIP film is able to selectively capture the template peptide and the corresponding target protein. In case of the peptide recognition, the MIP film can discriminate even the single amino acid mismatched sequences of the target peptide.