The invention provides a formula and preparation method of Gui powder for freshwater fish culture, and relates to the technical field of freshwater fish culture. The formula of the Gui powder for freshwater fish culture comprises magnesium, calcium, potassium, phosphorus, sulfur, carbon, zinc, sodium, folic acid, nicotinamide, calcium pantothenate, biotin, vitamin D3, vitamin E, vitamin K3, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C and vitamin A. According to the invention, by selecting the phosphorus component raw material, the stability of the properties of the selected phosphorus in the processing and using processes is ensured, radioactive elements are not generated, the safety of the raw material of the Gui powder is further ensured, and 10% of water is retained in the finished product of the Gui powder, so that the finished product of the Gui powder has better water melting performance, the Gui powder can uniformly act on the culture water area of freshwater fish, and in terms of the quality of the finished product, the dosage of the finished product of the Gui powder is small, the proportion is reasonable, and the function is relatively good.