As the human population grows, the demand for pure and germ-free water also increases simultaneously. Conventional microbial testing techniques are costly and time-consuming. The common bacterial pathogen that exists in almost all types of contaminated water, i.e., Escherichia coli (E. coli.) This typical bacterium is responsible for various human diseases like anemia, diarrhea, and sometimes death in serious cases. The detection of these bacteria in water samples is very important to overcome the aforesaid diseases. Here we are reporting a colorimetric method for the detection of E. coli. that is based on functionalized gold nanoparticles. Gold nanoparticles were synthesized as per standard protocol and characterized with the help of both instruments, i.e., a UV–vis spectrophotometer and a scanning electron microscope. In the presence of a bacterial pathogen (E. coli), a color change of the nanoparticle solution from red to blue takes place. The developed colorimetric method is reliable, reproducible, and instantly detects bacterial pathogens. The method was also tested for real water samples like RO water, tap water, and industrial water.