Tetraploid fish are important for mass production of triploids in polyploid breeding. In this study, we reported a novel protocol for artificial induction of tetraploidy by a combination of cold shock and hydrostatic pressure administered to gynogenetically developed eggs in Japanese flounder Paralichthys olivaceus. The induction was carried out by activating the eggs with UV-irradiated sperm of red sea bream Pagrus major, administering a cold shock (0°C, 5 to 45 min) 3 min after fertilization to inhibit second polar body exclusion, incubating the eggs for 60 min at 17°C, and treating them with a 650 kg/cm2 hydrostatics pressure shock for 6 min. We named the embryos gyno-tetraploids that developed from eggs after such treatments. The hatching rate of the gyno-tetraploids ranged from 20.99%±3.66% to 36.01%±2.79%, and the tetraploid rate ranged from 80.00% to 100.00%. All-maternal inheritance was verified using 6 high-recombination-rate microsatellite markers. This method successfully induced gyno-tetraploidy. The successful induction of gyno-tetraploidy lays the foundation for triploidization of new varieties with improved economic traits of interest that can benefit commercial culture.