The invention provides a heatproof lyoprotectant for a live vaccine against pseudorabies. The heatproof lyoprotectant comprises, by weight, 3 to 10 parts of gelatin, 1 to 5 parts of trehalose, 5 to 15 parts of sucrose, 0.1 to 2 parts bovine serum albumin, 1 to 8 parts of tryptone, 2 to 10 parts of enzyme-hydrolyzed casein, 1 to 5 parts of thiourea, 0.8 to 2 parts of L-monosodium glutamate, 0.1 to 3 parts of arginine, 0.5 to 5 parts of polyvinylpyrrolidone (PVP-K30) and 0.1 to 2 parts of mannitol. The invention also discloses a preparation method of the heatproof lyoprotectant and a lyophilized vaccine prepared from the heatproof lyoprotectant. When the heatproof lyoprotectant is used for protecting the vaccine and a specific lyophilization process is employed, lyophilization loss of viruses can be effectively reduced, the temperature tolerance of the viruses can be improved, and the vaccine can still maintain good physical properties and titer after long-term storage; i.e., the vaccine has stable characters and has the characteristics of heat resistance and long storage time.