Listeria monocytogenes is a psychrophilic bacterium that can grow at low temperatures. The growth of L. monocytogenes in refrigerated ready-to-eat food and frozen food processing equipment is a major food safety issue. Acidic electrolyzed water (AEW) is a new type of high-efficiency, safe and no-residue non-heat sterilization technology. AEW is used to inactivate L. monocytogenes in planktonic state, biofilm state, and frozen Flammulina velutipes samples under low temperature conditions. By detecting changes in the total number of colonies after AEW treatment, biofilm, biofilm structural parameters, and virulence gene expression, we made further analysis of sample tests, etc.. The results showed that when the electrolyte concentration was 0.10% and the treatment time was 1 min and 30 min, the total number of Listeria monocytogenes colonies and the biofilm clearance rate at 4℃ and 37℃ were extremely different. That was, with the same treatment time, AEW was more difficult to inactivate L. monocytogenes cultured at 4℃. Our findings may provide a theoretical basis for evaluating the safety of food and refrigerated food and food processing equipment in the cold chain. The use of antibacterial agents is more demanding.