The invention discloses a preparation method of a reactive gemini quaternary ammonium salt leather bactericide. The preparation method comprises the following three steps: first step, carrying out esterification reaction on bromoacetyl bromide and fatty alcohol to generate bromine acetate; second step, carrying out nucleophilic substitution on trans-1,4-dichloro-2-butene and diethylamine to generate trans-N,N,N',N'-tetraethyl-2-butene-1,4-diamine; and third step, carrying out quaterisation on products obtained from the first two steps to obtain a reactive gemini quaternary ammonium salt. The preparation method is easy to operate, the yield of products is high, the cost of raw materials is low, the practicality is high, and moreover, a method for synthesizing diamine intermediate in the second step provides a new thinking and new method for synthesis of the gemini quaternary ammonium salt. Owing to existing of prepared bactericide molecular structure ester groups, the sterilizing property is improved, and biodegradability can be good. In addition, owing to existing of active groups C=C in the structure, the chemical activity of the gemini quaternary ammonium salt is improved, the gemini quaternary ammonium salt can be further chemically bounded into skin collagen and leather coating resin materials, and the long-lasting sterilizing property can be achieved.