The invention provides a turtle feed in fish-turtle polyculture mode and a preparation method of the feed. The feed comprises white fish meal, red fish meal, alpha-starch, extruded soybean, vinegar residue powder, beer yeast, mineral additives, multi-vitamins, feed binder, calcium hydrogen phosphate, sodium dihydrogen phosphate, carnitine, betaine, bile acid, methionine, lysine, nucleotide, choline chloride, and salt. The preparation method comprises the following steps: fully mixing the ingredients with stirring to prepare a feed, then feeding, precleaning, mixing, deironing, crushing, conducting superfine grinding, and mixing. The feed disclosed herein has scientific and reasonable combination, enhances the immunity and contagious virus resistance of the turtle, guarantees the normal growth of the turtle, simultaneously reduces the discharge of nitrogen, phosphor and other pollutants, reduces the pollution to the culture water body, reduces drug dosage, achieves the requirements of ecological breeding, and obviously reduces the cost. The preparation method is scientific and reasonable. The invention has significant economic benefits and social benefits.