The invention discloses a quaternary ammonium salt disinfection solution, and a preparation method and application thereof. The quaternary ammonium salt disinfection solution comprises the following components: hexamethylenetetramine, benzalkonium bromide, benzalkonium chloride, polyhexamethylene guanidine, Tween 80, octadecyldimethylbenzyl chloride, isopropanol and sterile water. The invention further discloses a method for preparing the disinfection solution. According to the invention, hexamethylenetetramine, benzalkonium bromide, benzalkonium chloride, polyhexamethylene guanidine, octadecyldimethylbenzyl chloride and Tween 80 are combined together according to a specific mixing proportion, and the components have synergistic effects, so a variety of disadvantages of a single disinfectant variety or a simply-mixed disinfectant are overcome, and a variety of bacteria, fungi, spores and viruses can be thoroughly killed; meanwhile, the disinfection solution is safe to human body, is free of toxicity and stimulation, is convenient to use, can be directly used by being diluted with water, realizes one-step completion of disinfection and cleaning, has no corrosion to equipment and no harm on the environment, and is biodegradable.