In order to improve the survival rate of Litopenaeus vannamei during transportation with water after carbon dioxide (CO₂) anesthesia, the effects of CO₂ flow rates (anesthesia stage), soaking time (soaking stage), water type and salinity (recovery stage) on the survival rate were studied. The experiment set four factors:CO₂ flow rates (3 L/min, 5 L/min and 7 L/min), soaking time (0 min, 1 min and 5 min), water type (tap water, supernatant of aquaculture water and aquaculture water) and salinity (3, 7 and 11). The single factor experiment was carried out, and the L₉(3⁴) orthogonal experiment and verification experiment were carried out based on the single factor experimental results. The results showed that in the single factor experiment, the best CO₂ flow rates was 7 L/min, the best soaking time was 0 min, the best water type was tap water and the best salinity was 7. The results of orthogonal experiment of transportation within 2 hours showed that the primary and secondary relationship of the four factors was:soaking time > water type > salinity > CO₂ flow rates, and the best combination was CO₂ flow rates of 8 L/min, the water type of tap water, the salinity of 7 and the soaking time of 0 min. The results of orthogonal experiment of transportation within 4 hours showed that the primary and secondary relationship of the four factors was soaking time > CO₂ flow rates > water type > salinity, and the best combination was CO₂ flow rates of 7 L/min, the water type of tap water, the salinity of 7 and the soaking time of 0 min. The results of orthogonal experiment of transportation within 6 hours showed that the primary and secondary relationship of the four factors was soaking time > CO₂ flow rates > salinity > water type, and the best combination was CO₂ flow rates of 6 L/min, the water type of tap water, the salinity of 7 and the soaking time of 0 min. The experimental results show that the recovery rate of Litopenaeus vannamei under different transportation time conditions can be improved by adjusting the levels of four factors:CO₂ flow rates, soaking time, water type and salinity, which provides a new idea for shrimp keep-alive transportation.