In this paper, we aim to construct an evaluation method for bicycle-riding comfort using biological signals. In recent years, it has become important, in bicycle development companies (e.g., MIYATA CYCLE Co., Ltd.), to develop products by reflecting human sensibility in order to improve user satisfaction. In addition to conventional evaluation methods using acceleration, angular velocity, and questionnaires, we focus on the electromyogram (EMG) and the electroencephalogram (EEG) as new evaluation indexes. We analyze the EMG and the EEG to estimate the features of these biological signals and how they reflect changes in bicycle-riding comfort.