The behavioral and physiological responses of tilapia to air exposure after pure saturated carbon dioxide (CO₂) anesthesia were analyzed, and a potential application of live tilapia dehydrated transport was evaluated by comparing it with the tricaine methanesulfonate (MS-222) anesthesia and no anesthesia. Tilapia displayed about 1 min of hyperactivity, and the induction time of anesthetized was about 3 min during CO₂ anesthesia, both the behavioral analysis and plasma cortisol levels showed that tilapia were more sensitive and stressed to the CO₂ anesthesia than MS-222 anesthesia. During air exposure, both anesthesia groups had good sedative effects, and the survival time of unanesthetized group, CO₂ anesthesia group and MS-222 anesthesia group were 331, 489 and 528 min respectively, and the survival time and survival rates of the anesthetized tilapia were significantly increased. Considering the efficacy, ease of use, as well as safety, the results of this study showed that CO₂ was an ideal anesthetic to improve air exposure duration of tilapia, and carbon dioxide anesthesia of tilapia potentially can be translated into an industrial short-time air exposure procedure.