The invention discloses a protective agent for low-temperature preservation of asexual spores of antrodia cinnamomea and a protective agent using method. The protective agent can be used for the preservation of antrodia cinnamomea strains and inoculums, and comprises 10%-30% of glycerin, 5%-15% of trehalose, 2%-10% of cane sugar and 2%-10% of lactose according to weight in weight (w/w); normal saline or water is used as a solvent; the preservation temperature is -80 DEG C to 4 DEG C. The protective agent provided by the invention can be used for preserving the asexual spores of the antrodia cinnamomea for a long time (more than one year) at the low temperature of -80 DEG C to 4 DEG C, and keeping a relatively high germination rate and growth and metabolism activity of the asexual spores. The preserved asexual spores of the antrodia cinnamomea can serve as the antrodia cinnamomea strains to be activated, transferred and bred; conventional oblique planes and glycerin pipes are replaced; a new preservation method is provided for the preservation of laboratory antrodia cinnamomea strains, is long in preservation time and has a good effect; in addition, the preserved asexual spores of the antrodia cinnamomea can also serve as the inoculums so as to carry out antrodia cinnamomea liquid fermentation and solid fermentation cultivation, thus the time and cost of seed preparation are saved for antrodia cinnamomea fermentation production, and the production efficiency is improved.