Abstract: [Objective]The detection effect of equine Toxoplasma gondii infection with serum samples prepared by three different protocols, including 4 ℃ storage overnight, 37 ℃ incubation in water bath, and stationary incubation at room temperature, was assessed in this study. [Methods]The horse blood samples after natural coagulation were placed at 4 ℃, room temperature, and37 ℃ respectively, and the obtained serum samples were subjected to Toxoplasma gondii antibody detection by IHA method; the antibody positive serum samples were selected and placed at 4 ℃ and room temperature respectively, and the antibody titer were determined daily for fifteen consecutive days. [Results]No differences in volume, color and brightness of the serum samples prepared with the above three protocols were observed; the same results were obtained in Toxoplasma gondii antibody detection for the serum samples prepared by different protocols; during the fifteen days observation duration, the brightness and antibody titer of the antibody positive serum samples placed at both 4 ℃ and room temperature had no obvious change. [Conclusion]The serum samples prepared by 4 ℃ storage overnight, 37 ℃ incubation in water bath and stationary incubation at room temperature are all qualified to be used in detection of Toxoplasma gondii infection; serum samples storage at 4 ℃ and room temperature for fifteen days are still able to be used for Toxoplasma gondii antibody detection.