The invention belongs to field of biological treatment of nitrate pollution in groundwater, and particularly relates to a preparation method of sulfur autotrophic denitrification bacterium immobilizedparticles. The preparation method of the sulfur autotrophic denitrification bacterium immobilized particles comprises the following steps that S1, polyvinyl alcohol and xanthan gum are added into deionized water, agricultural waste and sulfide which are subjected to biofilm culturing are added, and thus a material A is formed; S2, boric acid and calcium chloride are molten in the deionized water,a sodium hydroxide solution is added to adjust the pH value, then a supernatant is taken, and thus a solution B is formed; and S3, the material A is taken to be dripped into the solution B to form embedded particles, and after a cross-linking reaction is conducted, the sulfur autotrophic denitrification bacterium immobilized particles are obtained. According to the preparation method of the sulfur autotrophic denitrification bacterium immobilized particles, the mechanical properties and salt stability of the sulfur autotrophic denitrification bacterium immobilized particles can be improved, the denitrification start time is shortened, the denitrification efficiency is improved, and meanwhile, a new idea is provided for secondary utilization of the agricultural waste.