A new bacteria microarray patterning technique is developed by patterning thick polycationic polymers on glass surface, which generates high-coverage and high-precision E. coli cell patterns. Cell immobilization efficiency is greatly improved, compared to conventional monolayer surface patterning approach. Cell viability tests show very low cytotoxicity of polyethyleneimine (PEI). This advancement should further accelerate biomedical and bacteriological researches in the micro scale.