The invention discloses an efficient induction method and a microsatellite molecular marker identification method of Nile tilapia pseudo-female fishes. Different doses of estrogen diethylstilbestrol (DES) are used for sex reversal processing of different groups of Nile tilapia fishes to establish the efficient induction method of the Nile tilapia pseudo-female fishes; and a newly-developed microsatellite molecular marker tlp-23-sd and a primer thereof are further used for identification of the Nile tilapia pseudo-female fishes to establish the microsatellite molecular marker rapid identification method of the pseudo-female fishes, wherein the microsatellite molecular marker tlp-23-sd is closely linked with the sex. The efficient induction method is high in pseudo-female fish induction efficiency, and the microsatellite molecular marker rapid identification method is rapid, accurate and reliable in identification, and can be used for the production and identification of the Nile tilapia pseudo-female fishes.