The invention relates to an o-phthalaldehyde disinfectant and a preparation method thereof. The o-phthalaldehyde disinfectant comprises the following components in percentage by weight: 0.2 to 1 percent of o-phthalaldehyde, 0.1 to 0.5 percent of salicylic acid, 0.5 to 1.5 percent of dodecyldimethylbenzylammonium chloride, 1 to 10 percent of ethanol, 0.1 to 1 percent of disodium hydrogen phosphate, 1 to 10 percent of ethanol, 0.1 to 1 percent of sodium dihydrogen phosphate, 0.1 to 0.5 percent of inhibitor, 0.01 to 0.1 percent of ethylene diamine tetraacetic acid and the balance of water. The method for preparing the disinfectant comprises the steps of: adding the o-phthalaldehyde into the ethanol according to the content and component, stirring and mixing the materials; adding water into the materials and stirring the mixture; and adding the dodecyldimethylbenzylammonium chloride, the disodium hydrogen phosphate, the sodium dihydrogen phosphate, the inhibitor and the ethylene diamine tetraacetic acid into the mixture in sequence and stirring the mixed solution to allow the added materials to dissolve to obtain the o-phthalaldehyde disinfectant. The disinfectant has the advantages of no pungent smell, safety, low toxicity, stable property and convenient use, can be used for high-level disinfection of the heat-labile medical equipment and is particularly applicable to an endoscope automatic washing and disinfecting machine to perform high-level disinfection on an endoscope.