The consumption of contaminated food may cause serious illnesses, and traditional methods to detect Escherichia coli are still associated with long waiting times and high costs given the necessity to transport samples to specialized laboratories. There is a need to develop new technologies that allow cheap, fast, and direct monitoring at the site of interest. Thus, in this work, we developed optical immunosensors for the selective detection of E. coli, based on liquid crystal technology, whose molecules can align in different manners depending on the boundary conditions (such as substrates) as well as the environment that they experience. Each glass substrate was functionalized with anti-E. coli antibody using cysteamine as an intermediate, and a vertical alignment was imposed on the liquid crystal molecules by using DMOAP during functionalization. The presence of bacteria disrupts the alignment of the liquid crystal molecules, changing the intensity of light emerging between cross polarizers, measured using a polarized optical microscope and a monochromator. It was possible to detect E. coli in suspensions in the concentration range from 2.8 cells/mL to 2.8×109 cells/mL. Selectivity was also evaluated, and the sensors were used to analyze contaminated water samples. A prototype was developed to allow faster, in-situ, and easier analysis avoiding bulky instruments.