The biosensor described here uses a novel aqueous-based nanoparticulate polyaniline (PANI), doped with dodecylbenzenesulfonic acid (DBSA). The nanoparticles were applied to a glassy carbon electrode surface by electrodeposition techniques, and horseradish peroxidase (HRP) was subsequently electrostatically adsorbed to the nanoparticle-modified surface. This biosensor format was demonstrated for H2O2 sensing. Electrodeposited polymer nanoparticles resulted in highly ordered conductive nanostructured films, which were examined by scanning electron microscopy (SEM), atomic force microscopy (AFM), profilometry and spectroelectrochemistry. The surface of the films were characterized by a uniform array of nanoparticulate PANI (nanoPANI/DBSA) nodules and were shown to have a thickness of 350 nm. Physical techniques have shown that the nanofilms possess properties which allow for uniform electrostatic adsorption of protein to take place. This effective biosensor format, exhibits higher signal-to-background ratios and shorter response times than previous PANI biosensor configurations.