The invention relates to a hydrogen peroxide and peracetic acid mixed disinfectant and a preparation method of the disinfectant. The disinfectant is prepared by the steps of mixing hydrogen peroxide, acetic acid and sulfuric acid at the room temperature to prepare mixture containing peracetic acid and hydrogen peroxide, feeding a stabilizer and a surface active agent into the mixture containing the peracetic acid and the hydrogen peroxide, and mixing. The mixed disinfectant comprises 3-9g/ 100g peracetic acid, 20-35g/ 100g hydrogen peroxide, 5-12g/ 100g acetic acid, 0.1-2g/ 100g sulfuric acid, 0.05-2g/ 100g stabilizer, 0.05-2g/ 100g surface active agent and the balance of water. The hydrogen peroxide and peracetic acid mixed disinfectant is good in stability, is capable of rapidly killing various microorganisms such as bacteria, mould, yeast, spores and the like, and is safe, non-toxic and free from secondary pollution.