Relating to the technical field of aquaculture, the invention provides a biological fish guano for aquaculture and a preparation method thereof. The biological fish guano comprises the following raw materials by weight: 40-60 parts of an organic fertilizer, 20-30 parts of an inorganic fertilizer, 5-12 parts of a trace element additive, 0.4-0.7 part of a vitamin additive, 10-20 parts of amino acid,1-3 parts of beneficial bacteria, 4-9 parts of a Chinese herbal medicine additive, 2-5 parts of sodium percarbonate and 1-4 parts of sodium humate. The preparation method includes: weighing the raw materials in proportion, crushing the organic fertilizer, the inorganic fertilizer and the Chinese herbal medicine additive, then performing sieving with a 280-300mesh sieve, then mixing the obtained substances with other raw materials uniformly, and performing packaging to obtain the biological fish guano. The biological fish guano organically combines the long-acting effect of the organic fertilizer, the quick-acting effect of the inorganic fertilizer and the synergistic effect of the biological fertilizer, and avoids the defects of high oxygen consumption, high pollution and the like of thetraditional fertilizer. The biological fish guano has the advantages of scientifically and reasonably proportioned raw materials, more comprehensive nutrition and better water solubility, and can enhance the disease resistance of cultured varieties, and improve the yield and quality of the cultured varieties.