The invention provides a preparation method of low-crystallinity high trans-1,4-polyisoprene. The method comprises the following steps: sequentially mixing isoprene, a cocatalyst, a main catalyst and a comonomer; and respectively carrying out pre-polymerization reaction and polymerization reaction, so as to obtain the low-crystallinity high trans-1,4-polyisoprene, wherein the main catalyst contains a spherical support and an active component and a phosphate compound; the active component and the phosphate compound are loaded on the spherical support; the spherical support is a magnesium chloride alcohol compound or a spherical silicon dioxide; the comonomer is butadiene, pentadiene or 2,3-dimethyl-1,3-butadiene; and the molar ratio of the comonomer to the isoprene in the polymerization reaction is 2%-30%. The invention provides a method for preparing low-crystallinity high trans-1,4-polyisoprene. The problems of polymer morphology control, polymer liquidity and the like in a bulk precipitation polymerization process are effectively solved.