The invention discloses a preparation method of thyroxine (T4) labelled horse radish peroxidase (HRP), which comprises the following steps that (1) T4 and HRP are dissolved in a dimethyl sulfoxide solution and a carbonate buffer solution respectively; a solution A and a solution B are prepared; a tri-n-butylamine solution is added to the solution A; an ice bath is performed; stirring is performed for certain time continuously; a solution C is obtained; an isobutyl chloroformate solution is added to the solution C; the stirring is performed for certain time at a room temperature; a solution D is obtained; the solution B is added to the solution D for coupled reaction; a reaction mixture E is obtained; (2) the reaction mixture E is subjected to dialysis treatment to remove micromolecular substances not participating in coupling in the reaction mixture; the buffer solution adopts a 0.01M phosphate buffer solution with pH (potential of hydrogen) of 7.4; a coupled substance is obtained. The method has the advantage that T4 labelled HRP which is simple in preparation method, short in production cycle and low in production cost, and is used for thyroxine immunology detection is provided.