Abstract: The method of the peripheral blood lym-phocytic cell culture and chromosome preparation of the tonguefish (Cynoglossus semilaevis Gnther) was established in this research. The conditions of the peripheral blood lymphocytic cell culture and chromosome preparation were well searched from culturing temperature and time, effect of PHA, ConA, Lipopolysaccharide as immune enhancers, treating time and concentration of colchicines and so on. The result showed that 20% FBS with MEM and LPS (final concentration 0.3 mg/mL) was the best blood lymphocytic cell medium. Cul-tivating for 72h, treating the cells with colchicines (final concentration 0.08 g/mL) for 3h before culture ending could get better chromosome samples. We also presented a comparison of several methods to identify the sex of the tonguefish, including the preparation of chromosome from cultured peripheral blood lym-phocytic cell, female-specific DNA marker, Physiological anatomy, gonadal biopsy and gonad cell culture. The first two methods could identify genetic sex of tonguefish exactly, while the other three methods could identify physical sex of tonguefish. However, there were big limitations of anatomical observation, Gonadal biopsy and gonadal cell culture. Preparation of blood lymphocyte chro-mosome and female-specific marker methods could guarantee in vivo identification applicable, and the accuracy was also remarkable. The method of chromosome preparation could also be used to identify super female individual fish, while the method of female specific marker could not achieve. In a word, this experiment enriched the methods of ge-netic sex identification of Gnther in vivo.