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The purpose of this study is to investigate the differences of different gender people's attention in physiological evaluation during serious games play. This study used the physical sensor, electroencephalogram (EEG), and a theoretical model of motivation (Keller’s ARCS model) to evaluate the attention of different gender people during serious game play.Results of this study reveal significant differences between male and female subjects' attention, such as the male subjects' attention level is always higher than female's throughout the testing process; it is different in changes of different gender subjects' attention level in the same serious game.