The invention provides a method for detecting the injury of the mitochondria of the sperms of fishes, which relates to an aquaculture technology and needs to provide a method for detecting the injury of the mitochondria of the sperms of fishes. In the invention, the sperms of the fishes with a certain number are collected. The method is characterized by comprising the following steps of: after washing a sperm specimen by using PBS, centrifuging by 300*g for 5min; and suspending by the PBS, precipitating the sperms, regulating the density of the sperms to be 1*106/ml, adding Rhl23, hatching for 10min in a water bath with the temperature of 37 DEG C, centrifuging, removing supernatant, washing by the PBS repeatedly, centrifuging, suspending by the PBS, adding PI and dyeing for 5min. The sperms with the normal mitochondria and the injured mitochondria are dyed by adopting a fluorescent double-dyeing method and are directly detected by using a flow type cell analyzer after being preprocessed, and the sperms with favorable mitochondrial membrane functions, the sperms with lost mitochondrial membrane functions and the sperms with necrosis can be quantitatively distinguished.