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This invention provides a method for producing single-cell protein using a mixed bacterial culture, comprising the following steps: performing primary seed culture on methanogenic bacteria, *Tricholoma materata*, and *Glucosinolate methyl-eating bacteria* to obtain primary seed solutions; mixing the primary seed culture solutions evenly to obtain a mixed bacterial solution; inoculating the obtained mixed bacterial solution into a secondary seed culture medium for cultivation to obtain a secondary seed solution; inoculating the obtained secondary seed solution into a fermentation medium for scale-up cultivation to obtain a fermentation broth; separating the solid and liquid components of the obtained fermentation broth, harvesting the bacterial cells, and obtaining single-cell protein. This method for producing single-cell protein using a mixed bacterial culture improves the utilization rate of the carbon source methane and the yield of single-cell protein.