We investigated the effects of different environmental factors, namely, culture time and temperature, pH, and NaCl concentration, on autoinducer (AI-2) activity and biofilm formation of the foodborne pathogen Listeria monocytogenes. The results revealed that during the logarithmic growth period, the AI-2 activity of L. monocytogenes was initially enhanced with an increase in cell density, although on reaching the stable growth period, the activity showed a subsequent declining trend. The highest values for growth, biofilm biomass, and AI-2 activity were recorded when L. monocytogenes was cultured in polystyrene culture plates at 37 ℃ for 24 h, which were 7.65-, 11.14-, and 4.40-fold higher than the values obtained at 4 ℃, respectively. Listeria monocytogenes showed similar growth performance in response to the addition of 0.5-9.0 wt.% NaCl, with AI-2 activity peaking at a NaCl mass fraction of 3.0% and a relative fluorescence intensity of 6.159. The lowest values of biofilm biomass and AI-2 activity were recorded at an NaCl mass fraction of 9.0%. Moreover, at pH value 5 and pH value 9, AI-2 activities were 0.11- and 0.13-fold compared to the control group (pH value 7.4), respectively, indicating a significant inhibition of activity at both low and high pH values. Collectively, these findings indicate that AI-2 activity and biofilm formation of L. monocytogenes are influenced by environmental factors. These findings will provide a reference for further research on the regulation of biofilm formation by the LuxS/AI-2 quorum sensing system and its quenching.