We report visible spectroelectrochemical (SEC) characterization of cytochrome c552 (cyt c552) in viable Geobacter sulfurreducens biofilms on tin-doped indium oxide (ITO) electrodes poised at 0.24 V vs.. SHE. G. sulfurreducens biofilms were grown in minimal medium with acetate as electron donor (turnover conditions), followed by 24 hours incubation in electron donor-depleted medium (non-turnover conditions). The electronic absorption spectra of G. sulfurreducens biofilms showed the lowest energy absorption band in the reduced state at 552 nm, which indicated excess of cyt c552 in the biofilm. The spectra under non-turnover conditions displayed gradual reduction of the cyt c552, following the step-wise decrease of electrode potential from 0.0 V to -0.6 V vs.. standard calomel electrode (SCE). The spectral changes were fully reversible in both positive and negative direction of the scan potential, with average midpoint potential value of -0.42 V vs.. SCE. Confocal microscopy analysis revealed that the thickness of biofilms under turnover conditions and non-turnover conditions was approximately 35 and 3.5 µm, respectively. This is the first study to observe the reversible redox conversion of cyt c552 in viable G. sulfurreducens biofilms.