[Purpose] This study aimed to examine the relationship between chronic ankle sprain instability and ultrasonography of the peroneus muscles during a single-leg standing task. [Participants and Methods] We examined nine college-aged students with a history of lateral ankle joint sprain with chronic ankle sprain instability scores less than 24. Participants underwent ultrasonographic measurement of the pennation angle and muscle thickness of the peroneal and gastrocnemius muscle groups of both legs. In addition, participants were evaluated for fluctuation by the root mean square calculated from accelerations in the anteroposterior, lateral-horizontal, and vertical directions during the single-leg standing position by affixing the accelerometer to their waist. Measurement results were compared between sprain and non-sprain sides. [Results] Ultrasonography revealed a significant reduction in the feathered pennation angle of the long peroneal muscle on the side of the sprain, but no other significant differences. Also, significant extension was observed on the side of the sprain in the anteroposterior and vertical directions during single-leg standing; however, no significant differences were found in the lateral-horizontal direction. [Conclusion] Participants with chronic ankle sprain instability exhibited greater fluctuation in the anteroposterior and vertical directions. Such fluctuations are believed to be compensatory in nature because the feathered horn of the long peroneal muscle is decreased, and pronation of the forefoot is difficult during one-leg standing.