The invention discloses a spray for disinfecting an operation room before operation as well as a preparation method and a using method of the spray, belonging to the field of traditional Chinese medicines. The spray comprises the following raw materials of effective components: spiny aralia, semen cuscutae, day lily, gaultheria yunnanensis, horseweed herb, ficus pumila, maire sophora root, fructus ulmi, szechuan melandium root, Chinese gall, gamboge, broad-leaved epiphyllum, yarrow, salvia splendens, rhizoma curculiginis and coix lachryma-jobi. According to the spray, the compatibility of the medicines is proper; the spray is in accordance with the theories of traditional Chinese medicine and pharmacy and modern medicine and pharmacology; the spray is capable of rapidly killing viruses, bacteria, funguses, spores and the like, so that the infection is reduced in the operation process, the harm of infection in the operation room to a patient is effectively prevented, and the safety of operation can be improved; furthermore, the spray is also used for disinfecting various appliances and environments, is free from adverse effect to the human body and is easy to use, low in price and remarkable in effect, and the spread of bacteria in the air can be avoided, so that the spray is widely applied in the operation room.