In view of the high cost and serious environmental pollution caused by the disposal of municipal solid waste incineration (MSWI) fly ash, a method for preparing unburned microbial MSWI fly ash brick, using the principle of microbial mineralization, is proposed. The solidification and stabilization of heavy metals in MSWI fly ash were achieved by adding microbial bacteria liquid to the MSWI fly ash brick consolidation body and using the principle of microbial induced carbonate deposition. In this paper, MSWI fly ash, Ca(OH)2, sand and microbial bacteria were used as raw materials, and the optimal MSWI fly ash content, microbial bacterial solution concentration and calcium ion concentration in nutrient solution for the preparation of microbial MSWI fly ash brick were explored through single factor experiment. The test results show that when the MSWI fly ash content is 40% (mass fraction), the OD600 value of the microbial bacterial solution is 0.60, and the Ca2+ concentration in the nutrient solution is 0.30 mol/L, the mechanical properties of the MSWI fly ash brick are the best. At this time, the dry density of the 100 mm×100 mm×50 mm unburned microbial MSWI fly ash brick is 1 937.40 kg/m3, the compressive strength reaches 33.90 MPa, and the heavy metal leaching concentration meets the limit requirement, realizing the resource utilization of MSWI fly ash.