Tilapia were subjected to cooling (CO, a stepwise reduction in temperature from 30 °C to 15 °C), anesthesia (AN, anesthetized by hyperoxic carbon dioxide), air exposure (AE, exposed to air) and cold tolerance (CT, in 15 °C water) treatments, and the physiological responses were determined after the treatments. CO followed by AN treatment for tilapia could meet the criteria of an ideal anesthetic. Fish were deeply sedated within 69 s, completely anesthetized within 276 s and recovered within 308 s without any mortality. The stress responses induced by the CO&AN treatment were mild, whereas they were consistently increased in the AE treatment. Furthermore, the AE treatment caused tissue damage. The AE duration was significantly improved by CO&AN treatment, and the survival time of the CO&AE, AN&AE and CO&AN&AE treatments were 313 min, 351 min and 561 min, respectively, in the laboratory experiments, whereas the survival rate of the CO&AN&AE treatment group after 240-min air exposure was 95.2% in the pilot test. It appeared that cooling followed by hyperoxic CO2 anesthesia would be suitable for handling tilapia in a short-time air exposure procedure.