The electrochemical redox-induced contact angle changes of hemoglobin droplets in the absence and presence of tetraheptylammonium-capped Fe3O4 nanoparticles have been explored by using in situ electrochemical contact angle measurements. The results indicate that the electrochemical redox process may lead to some structure changes of hemoglobin (Hb), which could further induce the hydrophobic-to-hydrophilic changes of the relative droplets. Our observations demonstrate that hemoglobin could self-assemble on the surface of the functionalized Fe3O4 nanoparticles as Hb–Fe3O4 nanocomposites, which may contribute to much more significant change of the electrochemical redox-induced contact angle values than that with free nano-Fe3O4. These results suggest that in situ electrochemical contact angle measurements could be readily applied as a new and convenient method to detect some specific biological process.