Over the last decade, the M13 bacteriophage has been used widely in various applications, such as sensors, bio-templating, and solar cells. The M13 colorimetric sensor was developed to detect toxic gases to protect the environment, human health, and national security. Recent developments in phage-based colorimetric sensor technologies have focused on improving the sensing characteristics, such as the sensitivity and selectivity on a large scale. On the other hand, few studies have examined precisely controllable micro-patterning techniques in phage-based self-assembly. This paper developed a color patterning technique through self-assembly of the M13 bacteriophages. The phage was self-assembled into a nanostructure through precise temperature control at the meniscus interface. Furthermore, barcode color patterns could be fabricated using self-assembled M13 bacteriophage on micrometer scale areas by manipulating the grooves on the SiO₂ surface. The color patterns exhibited color tunability based on the phage nano-bundles reactivity. Overall, the proposed color patterning technique is expected to be useful for preparing new color sensors and security patterns.