We developed a bio-microelectromechanical system (bio-MEMS) device by combining the dielectrophoresis (DEP) method and MEMS technology that to trap a single PC12D (rat phenocromocytoma cell) for cell pattern formation to measure the axonal extension function. Our device could trap PC12Ds to assign bicells, with designed distances between two cells of 100, 200, and . PC12Ds were injected into our bio-MEMS device using a micropipette. We observed the axonal extension using a time-lapse microscope to study the effect of the bicell distance on the axonal extension and connection ability. An alternative electric field (frequency: 10 MHz, voltage: ) was applied for the PC12D DEP patterning. To keep the patterned PC12Ds on the electrode holes, water was injected using a microsyringe to sweep out excess cells, which remained outside the holes. An average single PC12D pattern ratio of 89.3% was achieved. The axonal extension ability was affected by the cell distance. PC12D bicell distances of 100 and showed good connection ratios of 86.7% and 91.7%, respectively, where as a bicell distance of showed a low-connection ratio of about 40.0%. We confirmed the applicability of our bio-MEMS device to bicell patterning and evaluated the cell extension function.