In this study, using twelve novel microsatellite markers, we established a paternity test technology for Acanthopagrus latus based on a mixed family of A. latus including 112 parent individuals and 393 offspring individuals. Genetic diversity data demonstrated that there were 119 alleles observed and the mean number of alleles (N_a), observed heterozygosity (H_o), expected heterozygosity (H_e) and polymorphism information content (PIC) were 9.91, 0.651, 0.661, 0.621, respectively. Paternity analysis showed that the combined probability of exclusion reached 99.58% when both parents were unknown (confidence level, 95%) by 12 loci. And, the combined probability of exclusion reached 99.1% when both parents were unknown (confidence level, 95%) by 8 loci. Consequently, the 8 high polymorphic microsatellite markers (AL49, AL37, AL01, AL20, AL14, AL18, AL15 and AL51) made up the system of paternity test for A. latus. The cumulative exclusion rate of their parents was 99.1% without parents' gender by 8 loci. The rate of assignment success for the real data set was only 89.31%. In conclusion, the combination of this novel 12 microsatellite markers in our study provided highly effective testing tools for the paternity identification, population breeding and molecular-assisted family management in A. Latus.