Objective To study the thermal inactivation of Listeria monocytogenes in different substrates at different temperatures, and to explore the effect of sous-vide on the reduction and elimination of Listeria monocytogenes in different substrates. Methods Five strains of Listeria monocytogenes were artificially contaminated with freeze-dried bacteria powder in normal saline, MH broth, lean pork and streaky pork. The survival rules of the bacteria in normal saline, MH broth and sous-vide packed lean pork and streaky pork were measured at 55℃, 60℃ and 63℃. The D and Z values of different strains were calculated under different conditions. The inactivation of Listeria monocytogenes in different substrates at different temperatures was analyzed. Results D value and Z value of Listeria monocytogenes in pork were significantly higher than those in normal saline and MH broth (P < 0.01), and D value in streaky pork was significantly higher than that in lean pork (P < 0.01). At 55℃, the time required for artificially contaminated Listeria monocytogenes to decrease 6log in sous-vide packed lean pork and streaky pork matrix was about 3-4 hours, while the time was 30-40 minutes and 10-20 minutes at 60℃ and 63℃, respectively. Conclusion Under the conditions of sous-vide, food substrate and genetic basis of strains have significant effects on the killing effect of Listeria monocytogenes. When cooking at below 60℃, the cooking time should be extended to ensure the microbial safety of cooked food.