The invention discloses a preparation method of 1,3,5-hexatriene. The 1,3,5-hexatriene is obtained by taking an inert electrode as a working electrode and a counter electrode, and performing electrolysis by taking a solution of a muconic acid solution in protic or aprotic solvent as an electrolyte. According to the preparation method of the 1,3,5-hexatriene, an electrochemical method is firstly adopted by the invention to prepared the 1,3,5-hexatriene; muconic acid is a renewable green raw material; an aqueous solution system can be completely adopted; the obtained 1,3,5-hexatriene is separated naturally, and the product purity is high.