The role of sigB, a major transcriptional regulator of stress response genes of Listeria monocytogenes, was assessed in biofilm formation of wild type strain (WaX12) and sigB deletion mutant (WaX12-ΔsigB) as affected by temperatures (4℃, 15℃, 25℃ and 37℃), pH values (4, 5, 6 and 7) and NaCl concentrations (0.5%, 2.5%, 4.5% and 6.5%). Results showed that the biofilm biomass was significantly reduced in L. monocytogenes mutant lacking sigB (P<0.05). According to the coefficient of variation analysis, all three of the culture conditions tested (i.e., temperature, pH and NaCl concentration) appeared to affect the strain variability of the biofilm formation of WaX12 and WaX12-ΔsigB, and the increase in the strain variability of biofilm formation caused by temperature is much greater than that caused by NaCl or pH. The ability of forming biofilm by WaX12-ΔsigB was more vulnerable to the effects of external culture conditions. There was a significant difference of biofilm biomass between WaX12 and WaX12-ΔsigB at 37℃, pH 6 and 2.5% NaCl, respectively, so the further research was observed under these culture conditions. Notably, results showed that the relative amounts of total polysaccharides and extracellular proteins were much lower in WaX12-ΔsigB biofilm (P<0.05). However, there was little influence on live cells. In conclusion, this study could provide a scientific basis for further investigation of the important role of sigB in L. monocytogenes biofilm formation.