The invention discloses a preparation method of carbon-based biological bacterial fertilizer. The fertilizer is prepared from, by weight, 20 parts of sheep manure organic fertilizer, 10 parts of biomass charcoal, 15 parts of potassium fulvic acid, 10 parts of bacillus subtilis culture substances, 10 parts of aspergillus awamori culture substances, 10 parts of bentonite, 8 parts of nitrogen-phosphorus-potassium, 5 parts of polyglutamic acid, 3 parts of potassium bacteria agent and the balance water. The preparation method comprises the following steps that S1, by weight, potassium fulvic acid,bacillus subtilis culture substances, aspergillus awamori culture substances, nitrogen-phosphorus-potassium, bentonite and water are added into a stirrer, stirring and mixing are conducted for 5 min at the rotation speed of 60 r/min, and a mixture A is prepared; the sheep manure organic fertilizer and biomass charcoal are used as a matrix and added into the mixture A for secondary fermentation. The preparation method is reasonable in design, the preparation technology is simple, the preparation cost is low, the survival rate and propagation speed of microorganisms are increased, a substrate required by survival of the microorganisms is improved and optimized, and the method is worthy of popularization.