Purple membrane (PM) monolayers were deposited on hydrophilic or hydrophobic silicon substrates through the Langmuir-Blodgett LB) technique. The photovoltaic features and interfacial charge separation of p-Si/PM/indium tin oxide (ITO) heterostructure were studied by surface photovoltage spectroscopy ASPS). The different photovoltaic response values were obtained due to the nonrandom orientation of PM in the LB films on the hydrophilic versus hydrophobic silicon substrates. The photovoltaic response value versus external potential of the p-Si/cytoplasmic-extracellular/ITO heterostructure showed an obviously rectifying behavior. Compared with the p-Si/ITO heterostructure, the response value of SPS increased more rapidly in the case of the positive external bias.