The invention discloses a method for preparing a bee venom injection, which comprises the following steps of: (a) weighing bee venom, water for injection and octanol in a mass-to-volume ratio of 1:(20-30):(5-7); (b) dissolving the bee venom in the water for injection and then adding the octanol for extraction; separating extract liquor by using a sephadex column; and (c) freezing the bee venom for 1-2h at the temperature ranging from -10 DEG C to -15 DEG C; diluting the frozen bee venom solution to an specific amount with a prepared buffer solution; carrying out ultrafiltration by using ultrafiltration equipment, adding 0.05-0.1% of glycine, stirring, carrying out rough filtration and fine filtration and finally packaging. The bee venom injection prepared by the method has the advantage of stable quantity and high percent of pass.