This study aims to investigate the relationship between ergonomic comfort and the provision of accessibility features for mobility-impaired persons in the use of public transportation systems in Korea. To deal with mobility issues in a public transportation environment, we chose the low-floor bus as our research subject because it is the representative of a barrier-free design solution. We collected data via video observation, which can overcome the limitations of traditional accessibility-focused studies and laboratory studies by providing data on real usage patterns. We developed a framework to analyze the gathered data which includes user, space, tools, activities, and context. As a result, we observed existing difficulties of mobility-impaired persons in terms of moving and supporting their bodies on the bus, despite the application of accessibility features. Furthermore, the design only for accessibility of mobility-disabled persons can cause unexpected predicaments for the passengers who do not have physical handicaps. Drawing on these findings, we concluded that the concept of universal design in public transportation environments must be introduced in Korea. This study suggests video observation as a useful methodology for collecting data in dynamic environments. Additionally, our study is expected to contribute to how the concept of universal design can be implemented, and stimulate issues for ergonomic research based on our behavior pattern analysis.