The invention discloses an anti-stress agent for relieving transportation stress before fowl slaughtering, and a preparation method thereof. The anti-stress agent is prepared from the components in percentage by weight: 20 to 50 percent of glucose, 5 to 20 percent of guanidinoacetic acid glycine, 5 to 20 percent of glutamine, 5 to 13 percent of theanine, 5 to 10 percent of glycine betaine, 5 to 10percent of L-ascorbic acid, 1 to 5 percent of tannin, 1 to 4 percent of potassium citrate, 0 to 5 percent of sodium propionate, 0 to 5 percent of sodium diacetate, 0 to 5 percent of lactic acid, 1 to3 percent of potassium chloride, and 0 to 2 percent of sodium bicarbonate. 0.01 to 3 percent by weight of anti-stress agent is added into drinking water for fowl. The fowl drinks the anti-stress agent 48 hours before being loaded and 2 hours after being unloaded, and does not drink during the transportation process. After the fowl drinks the anti-stress agent, the moisture content of chicken canbe remarkably improved, the comprehensive stress reaction before slaughtering is reduced, the weight loss is reduced, and the economic loss is reduced.