The invention discloses a preparation method of disinfect and sterile spray for preventing bouine mastitis. The preparation method comprises the following steps of: A, taking 70-90 parts by weight of radix sophorae flavescentis, adding 280-360 parts by weight of deionized water, decocting for 50-60 minutes, filtrating, and concentrating till the relative density is 1.03-1.05 at a temperature of 60 DEG C; B, adding 520-640 parts by weight of deionized water into a dissolving kettle, heating to 40-50 DEG C, then adding 7-8 parts by weight of chlorhexidine gluconate, 60-80 parts by weight of glycerin, 15-20 parts by weight of menthol, and 240-340 parts by weight of ethanol and evenly mixing; and C, mixing a concentrated solution in the step A and a mixed solution in the step B, and adding 1-2 parts by weight of food brilliant blue and evenly stirring; and canning, and sealing to obtain the disinfect and sterile spray after 260-360 parts by weight of dimethyl ether is charged. An application method of the disinfect and sterile spray comprises the following steps of: vertically and directionally spraying the disinfect and sterile spray on the places which are 1cm far away from nipples below the nipples after milking each time, and covering liquid medicine on the nipples.