The invention relates to a preparing method of a mother culture special for production of Cordyceps militaris. The preparing method includes: adding, according to a certain ratio, nutrient solution containing cane sugar, monopotassium phosphate and magnesium sulfate to wheat bran and millet, adjusting pH to obtain a medium, charging the medium in a flask, performing sterilizing at 121 DEG C for 30 min, in aseptic conditions, inoculating a spawn of Cordyceps militaris, performing culturing constantly at 18 DEG C to 25 DEG C for 7 to 14 days to obtain the mother culture. After the mother culture is added a certain amount of aseptic water, the mother culture is inoculated to a fermentation tank or solid medium, and the mycelia and fruiting bodies of Cordyceps militaris can be produced on a large scale. The preparing method has the advantages that the step amplified cultivation process is omitted, the production cycle is shortened by greater than one third, the cost of seed step amplification equipment is reduced, the production cost is decreased by greater than one third, inoculating is simple, less contamination occurs, the risk of strain degeneration is reduced, and the mycelia and fruiting bodies of Cordyceps militaris can be industrially produced.