The invention provides a cynoglossus semilaevis microsatellite marker triplex PCR parentage identification primer and method. The method comprises the steps of marking three pairs of microsatellite primers by using two kinds of fluorescence, and distinguishing the microsatellite primers according to fluorescence colors and fragment sizes of PCR amplification products of the microsatellite primers; eliminating the possibility that the three pairs of microsatellite primers form dimers in a PCR reaction process by using primer design software; combining the three pairs of microsatellite primers into the same PCR reaction system; optimizing the PCR reaction system, and establishing parameters and a PCR reaction program through which the three pairs of microsatellite primers can simultaneously amplify in the same PCR reaction system; performing a molecular signature analysis on PCR products through an ABI 3130 or a similar genetic analyzer, judging the products with the same fluorescent marker through the segment sizes, and distinguishing different parentages of cynoglossus semilaevis or recognizing and identifying between individuals and between parents and children. According to the cynoglossus semilaevis microsatellite marker triplex PCR parentage identification primer and method, specific enhancement and releasing population of the cynoglossus semilaevis can be traced, and distribution, zoology, quantity of resources and releasing effect of the released cynoglossus semilaevis can be evaluated.