The invention relates to preparation and application of a sterilization composition containing various effective sterilization components. The sterilization composition contains a guanidine compound, a quarternary ammonium compound, 2-bromine-2-nitro-1,3-propanediol and ethanol, is diluted within a range of 50-1000 times, has the average killing rate of 99.99-99.9999 percent to gonites, bacterialspores, mycobacteria, viruses, fungi and spores thereof, can be used for the public health field, the food processing industry, the medical and health field, the livestock and poultry industry, the aquaculture industry and sterilization treatment on indoor air and in-vehicle air and can also be used for the port field, in particular to ship ballast water treatment, container treatment, industrialcirculating water treatment, swimming pool water treatment, hand and skin mucosa treatment, industrial wastewater treatment, hospital wastewater treatment and antiseptic treatment of a building material.