The invention discloses a preparation method of a bio-organic fertilizer which is prepared from nitrate mud, animal waste, flint clay, a rapeseed cake, phosphorus powder, plant ash and biogas fluid via combination and fermentation. The preparation process comprises the following steps: respectively airing the nitrate mud, the flint clay, the phosphorus powder, the rapeseed cake, the plant ash and the animal waste until the water content of the materials is lower than 12%, then respectively smashing the materials, mixing the materials and stirring uniformly to obtain a mixed material, mixing the biogas fluid with the mixed material to stir uniformly and adjusting the water content of the mixed material to 55-65%, and carrying out pile fermentation. The bio-organic fertilizer is wide in raw material source, low in cost, simple in preparation process, low in prepared product cost, comprehensive in nutrient element and good in fertilizer effect, and has the properties of increasing the organic matters of the soil, improving the soil structure, promoting the microbial activities of the soil, strengthening the fertilizer maintenance and fertilizer supply of the soil, increasing the crop output and improving the agricultural product quality.