In this study, we examined the fundamental surface characteristics of a polyserotonin (pST) film, which is attractive as a bioelectrical and biocompatible interface of biosensors. The pST film can easily be modified on electrode materials such as Au by self-polymerization and electropolymerization. By a simple cytotoxicity test using nonadhesive living cells, we found that the pST film is biocompatible for culturing cells on it. This finding is also supported by the fact that the surface tension of the pST film is moderate for protein adsorptions. The pST film is thinner and smoother than a poly-dopamine film, the chemical structure of which is similar to that of the pST film, depending on the polymerization time, cycle, and temperature; thus, ST as the main monomer can facilitate the precise control of the thickness and roughness of functional polymer membranes on the nanometer order. In addition, the pST film is useful as a relatively insulative interface for preventing interfering species from approaching electrode surfaces without their nonspecific adsorption, depending on the surface charges of the pST film in solutions of different pHs. The formation of the pST film self-polymerized on electrode materials is derived from the adsorption of pST nanoparticles formed by oxidative polymerization under basic conditions; therefore, the process of pST film formation should be considered in the functionalization of the pST film as a bioelectrical interface that allows biomolecular recognition (e.g., molecularly imprinted polymer membrane) for its application to wearable and biocompatible biosensors.