Abstract Scanning surface potential microscopy was applied to detect the photo-voltaic response of Langmuir–Blodgett (LB) monolayer assemblies containing an amphiphilic A–S–D triad molecule (ASD) with two different concentrations. The triad acts as a charge separation unit in the same way as the photosynthetic reaction center. In addition, the photo-induced multistep electron transfer systems can be organized in the LB monolayers: first electrons and holes are separated across the monolayer through the ASD triads upon photo-excitation and then the resultant electrons and holes are further separated by lateral diffusion among the concentrated A and the D moieties, respectively. The change in surface potential was clearly observed on an LB assembly with the high ASD concentration in a mixed ASD monolayer with ω-tricosenoic acid (T) (the molar ratio of ASD:T=1:5), while the photo-response could not be observed on an LB monolayer with the low ASD concentration (the molar ratio of ASD:T=1:30). It was interesting to note that the average surface concentration of ASD in the latter diluted monolayer was decreased only to 1 6 of that of the former concentrated monolayer. If the photo-response depended linearly on the ASD concentration, the surface potential would be readily detected in the latter monolayer. The nonlinearity can be attributed to the effect of succeeding lateral diffusion of the separated charges among the ASD triads. To clarify the effect of the lateral diffusion, the other type of LB assemblies, i.e., A/A–S–D double layers, was fabricated by alternate deposition of an additional acceptor (A) layer and the ASD layer. In the A/A–S–D assemblies, significant photo-induced surface potential change was observed with an A layer of a high A concentration (A:T=1:2) even when the ASD concentration was low in the ASD layer (ASD:T=1:30). This result supported an idea that the additional A layer enhanced lateral electron diffusion and resulted in electron accumulation in A layer and hole accumulation in the diluted ASD layer in the A/A–S–D system.