In this paper, Bacillus subtilis spores were treated with 0.8 g/L and 1.6 g/L tea polyphenols at different temperatures, and the inactivating effect of tea polyphenols combined with heat treatments on Bacillus subtilis spores was determined by plate counting method. The effect of tea polyphenol combined with heat on the energy metabolism of Bacillus subtilis was analyzed by measuring the activity of adenosine triphosphate (ATP) enzymes and tricarboxylic acid metabolizing enzymes (succinate dehydrogenase (SDH) and malate dehydrogenase (MDH)). The results showed that tea polyphenols combined with heat had a significant synergistic effect on Bacillus subtilis spores, and the inactivating effect was more pronounced as the concentration and temperature increased. After 1.6 g/L tea polyphenol combined with 100 °C treatment of spores, the total number of colonies decreased by 2.45 lg(CFU/mL), nucleic acid leakage and conductivity increased significantly (P < 0.05), OD260nm increased from 0.05 to 1.08, and conductivity increased from 6.61 ms/cm to 71.46 ms/cm, indicating that the treatment could disrupt the cell membrane of Bacillus subtilis spores. Scanning electron microscopy revealed that the morphology of Bacillus subtilis spores subjected to the tea polyphenol combined with heat treatment was wrinkled and depressed, and their ATP, SDH and MDH activities were significantly reduced (P < 0.05), as shown by a decrease of 0.16 U/mg in ATPase activity, 5.3 U/mg in SDH enzyme activity and 18.7 U/mg in MDH enzyme activity, indicating that the tea polyphenol combined with heat treatment could well disrupt the energy metabolism of Bacillus subtilis spores. In conclusion, tea polyphenol combined with heat treatment can disrupt the cell structure of Bacillus subtilis spores, and at the same time inhibit the activity of related enzymes in the respiratory metabolic pathway, which affects the normal growth of the bacterium and eventually leads to its death.