The invention discloses biological enzyme fish guano and a preparation method thereof, relating to the technical field of biological fish guano and preparation thereof. The biological enzyme fish guano comprises the following components: 300 parts of oil cake, 300 parts of chicken dung, 100 parts of rice bran, 100 parts of bone meal, 100 parts of shale, 50 parts of calcium superphosphate, 50 parts of urea, 10 parts of enzyme expansion bacteria, 6 parts of brown sugar and 1 parts of trace elements. On one hand, the biological fish guano can be used for killing harmful bacteria in a fish pond, inhibiting the propagation and growth of pathogenic bacteria in a water body, reducing diseases; and on the other hand, the biological fish guano can be used for breeding beneficial algae in an oriented way, supplying natural fry baits, recovering physical conditions of fishes as soon as possible, and simultaneously improving concentration of water in the pond, has a heat preservation effect and can effectively prevent the huge influence of external changeable weathers, such as the unfavorable influence of cold spell in later spring to the fries. Fishes cultivated with the biological enzyme fish guano are natural and ecological, and have long freshness retaining period, natural taste, wild forms, high yield and delicious flesh.