Since neurotransmitters play critical roles in nervous systems, the imaging techniques that can visualize them are needed. This communication describes the development of a method for simultaneous imaging of dopamine (DA) and glutamate (Glu), typical neurotransmitters, using enzyme‐modified large‐scale integration (LSI)‐based amperometric devices, referred to “Bio‐LSI”. The DA was detected by using direct electrochemical oxidation while Glu was measured by using an indirect electrochemical reaction involving glutamate oxidase (GluOx)/horseradish peroxidase (HRP) and osmium (Os) polymer‐modified electrodes. The Os‐HRP polymer was modified on designated sensor electrodes through electrodeposition. The selective and simultaneous imaging of neurotransmitters such as DA and Glu was achieved by applying the enzyme‐modified Bio‐LSI.