The invention relates to a method for enriching and purifying microorganisms in flue-cured tobacco. The method comprises the following steps: soaking, oscillating and washing the flue-cured tobacco in a dispersing agent, removing tobacco residue by using a screening mesh, and preparing tobacco microorganism suspension; and filtering the tobacco microorganism suspension by using filter cloth, kieselguhr and a microfiltration membrane at reduced pressure, washing by using a dispersing agent, and collecting filter cakes on the filter cloth, the kieselguhr and the microfiltration membrane to obtain the enriched and purified microorganisms in the flue-cured tobacco. By the method, the microorganisms in the flue-cured tobacco are subjected to graded filtration enrichment and multiple-washing purification, the steps are simple, the method is easy to implement, the enrichment efficiency is high, and the method is suitable for enriching and purifying microorganisms in flue-cured tobacco in any sample size, and is particularly suitable for enriching and purifying microorganisms in flue-cured tobacco in large sample size.