The invention discloses a preparation method of modified biochar immobilized petroleum degrading bacterium microspheres, which comprises the following steps: taking reed biomass powder, carbonizing, and activating in CO2 atmosphere to obtain a modified biochar carrier; placing the modified biochar carrier and a petroleum degrading bacterium solution in an immobilized culture medium for mixed adsorption to obtain the modified biochar adsorbing the microbial inoculum; a chitosan solution is used as an embedding agent to embed the modified biochar adsorbing the microbial inoculum, and the modified biochar immobilized petroleum degrading bacterium microspheres are obtained. The reed biochar is modified in a CO2 activation mode, the specific surface area and the pore volume of the immobilized carrier are effectively increased, chitosan is adopted as an embedding material, the degradation efficiency of petroleum degrading bacteria on petroleum is effectively improved, waste reeds are recycled, the problem of constructed wetland waste disposal can be solved, and the method is suitable for industrial production. The resource utilization of wastes is realized.