Microbes have been used extensively in various fields of researches and industries but has not been used widely for microfluidic biosensor applications because it is difficult to immobilize properly to a small space. Therefore, we developed a microbial immobilization method for microfluidic devices using single-walled nanotubes and dielectrophoretic force. Single-walled nanotubes and Escherichia coli were aligned between two cantilever electrodes by a positive dielectrophoretic force resulting in a film of single-walled nanotubes with attached Escherichia coli. The optimal condition of film formation without a cell lysis was investigated. Diameter of single-walled nanotubes and electric field (intensity and duration of application) had an effect on the cell viability. On the other hand, the cell concentration of the suspension did not affect the cell viability. Paraoxon was detected using single-walled nanotubes film with attached Escherichia coli that expressed organophosphorus hydrolase. This film which is suspended from the substrate showed faster response time than sensors that are not suspended from the substrate.