A fragment cDNA of parvalbumin gene has been cloned, which expressed differently between pre-metamorphosis and pro-metamorphosis stages of Japanese flounder, Paralichthys olivaceus. For the requirement of further research on the role of parvalbumin in metamorphosis stage, it is necessary to clone the full-length cDNA of parvalbumin gene. Here we constructed a head tissue full-length cDNA library of pro-metamorphosing flounder （23 dph, day post hatch） using SMARTTM technique. The titer of cDNA library was estimated as 1.2 ? 10^6 PFU?mL^-1, which was about 1000 times coverage of gene expressional profile, and the inserted fragments in the library are about 1000 bp length. Through two-runs anchor-PCR with target gene specific primers and λ TripIEx2 primers, we obtained a full-length cDNA of parvalbumin gene from the cDNA library. The full-length cDNA of parvalbumin gene consisted of 603 bp nucleofides, and encodes 109 amino acids with two EF-hand calcium-binding domains： DQDGSGFIEEEL （52- 64） and DSDGDGKIGVEEF（91 - 103）, and an ATP/GTP-binding site motif A （P-loop）： ACGLAGKS（ 33- 40）. The structure of the sequence shows the parvalbumin is a typical calcium-binding protein. Further analysis with homologue sequence comparison shows the flounder parvalbumin is a conserved protein, which shares 61.5% - 68.8% similarity with other teleosts, such as 61.5% with channel catfish lctalurus punctatus, 68.8 % with zebrafish Danio rerio. The phylogenetic analysis shows the closest relationship existed between parvalbumins of Japanese flounder and cod Theragra chalcogramma. Parvalbumin plays biological role of relaxation of muscle after contraction by soaking free calcium in cell. The recent researches show the parvalbumin gene expressed differentially in GABA neurons. However, it is unclear about the role of parvalbumin in metamorphosing flatfish, including Japanese flounder. The results reported here should be helpful for the lhture work such ~s gene expression location, gene function research.