We proposed a new bacteria patterning method on the restricted region of microbeads, using the submerged property of polystyrene microbeads on various concentrations of agarose gel. Moreover, we fabricated a bacterial microrobot using attenuated Salmonella typhimurium through the new patterning methods. We controlled the submerged degree of polystyrene microbeads through the regulation of the hardness of the agarose gel. The polystyrene microbeads on agarose gel were transferred onto a poly-dimethylsiloxane (PDMS) surface for easy manipulation of the microbeads. Then, we treated the polystyrene microbeads on the PDMS surface with antibacterial adherent factors, such as O2 plasma and bovine serum albumin (BSA). The Salmonella typhimurium was attached to the entire surface of the untreated polystyrene microbeads, whereas Salmonella typhimurium were only attached to the restricted surface region of the treated polystyrene microbeads through the proposed patterning method. The bacteria-attached microbeads gain motility by the propulsion of the attached bacteria, and the selective-bacteria-attached microbeads showed enhanced motility. Compared with whole-bacteria-attached polystyrene microbeads (1.74 ± 1.62 μm/s), the selective bacteria-attached polystyrene microbeads, using O2 plasma and BSA, showed 9.18 ± 1.88 μm/s and 14.65 ± 8.66 μm/s faster moving velocities, respectively. Through the results, we expected that the proposed patterning methodology of microbeads could contribute to the development of biomedical bacterial microrobots.