Abstract Organic monolayers are extensively used in various applications to modify substrate surfaces. However, their characterization remains challenging owing to their small thickness. Optical visualization techniques using antireflective (AR) substrates are particularly effective for patterned monolayers. In this study, we demonstrate the visualization of patterned alkanethiol monolayers on an AR multilayer substrate produced through microcontact printing methods. The AR substrate was designed to achieve near-zero reflectance at ~540 nm. The formation of the octadecanethiol monolayer caused a shift of approximately 2 nm toward longer wavelengths in the reflection spectrum. This shift was detected as a color difference by the digital camera attached to a reflected-light microscope. The contrast of the imaged pattern was enhanced by over 15% using a narrow band-pass filter at 530 or 550 nm. Consequently, line patterns with a width of approximately 6 μm were easily photographed using a commercially available color camera in a standard setup.