An optical brain-machine interface (O-BMI) system using calcium imaging has various advantages such as high resolution, a comprehensive view of large neural populations, abilities such as long-term stable recording, and applicability to freely behaving animals in neuroscience research. The present study developed an ergonomic user interface (UI) design, based on a use scenario for an O-BMI system that can be used for the acquisition and processing of calcium imaging in freely behaving rodents. The UI design was developed in three steps: (1) identification of design and function requirements of users, (2) establishment of a use scenario, and (3) development of a UI prototype. The UI design requirements were identified by a literature review, a benchmark of existing systems, and a focus group interview with five neuroscience researchers. Then, the use scenario was developed for tasks of data acquisition, feature extraction, and neural decoding for offline and online processing by considering the sequences of operations and needs of users. Lastly, a digital prototype incorporating an information architecture, graphic user interfaces, and simulated functions was fabricated. A usability test was conducted with five neuroscientists (work experience = 3.4 ± 1.1 years) and five ergonomic experts (work experience = 3.6 ± 2.7 years) to compare the digital prototypes with four existing systems (Miniscope, nVista, Mosaic, and Suite2p). The usability testing results showed that the ergonomic UI design was significantly preferred to the UI designs of the existing systems by reducing the task completion time by 10.1% to 70.2% on average, the scan path length by 14.4% to 88.7%, and perceived workload by 12.2% to 37.9%, increasing satisfaction by 11.3% to 74.3% in data acquisition and signal-extraction tasks. The present study demonstrates the significance of the user-centered design approach in the development of a system for neuroscience research. Further research is needed to validate the usability test results of the UI prototype as a corresponding real system is implemented.