The Bacillus subtilis spores were treated with High-pressure thermal sterilization combined with mass fraction fraction of 0.5%， 1%， and 2% tween 80 on Bacillus subtilis. The effect of tween 80 solution treatment on the survival of spore colonies was determined by plate counting， The flow cytometry， colorimetric method， ATP enzyme kit， Fourier transform infrared spectroscopy was used to analyse the effect of tween 80 solution on spore inner membrane permeability， DPA (2，6-pyriddiarboxylic acid) release， spore Na+/K+-ATP， Ca2+/Mg2-ATP enzyme kit and and stability of protein secondary structure. The results showed that tween 80 could weaken the inactivating effect of HPTS on Bacillus subtilis and exert a protective effect on spores. When the tween 80 mass fraction was 0.5%， combining the low pressure heat (200 MPa， 75 ℃)， the maximum survival of spores could reach 5.3 lg (CFU/mL). It was found that protection of the inner membrane permeability， DPA， ATP enzyme activity， and the secondary structural stability of the spore protein. Kinetic studies found that the spore sterilization effect was positively proportional to the treatment time， and the longer the treatment time， the stronger the spore inactivating effect. The sterilization process of HPTS combined with tween 80 complied with the Weibull model， and the parameter b and n (n<1) values of the Weibull model decreased after the addition of tween 80. As the treatment time continued， the temperature and pressure sensitive spores were quickly inactivated， while some super-deep dormant spores were survived.