Flatfish larvae metamorphose from bilaterally symmetrical larvae into asymmetrically shaped juveniles with two eyes in ocular side, and the ocular side pectoral fin is larger than the one on blind side. Many members of Tbx family take part in many organogenesis processes, including heart, limb and eye. However, the relationship between Tbx gene and flatfish larvae metamorphosis has not been reported. We used scanning electron microscopy (SEM) and bone/cartilage staining method to observe the morphology in Paralichthys olivaceus pre-metamorphosis stage (2 dph) and pro-metamorphosis stage (26 dph) larvae. The results indicate that pectoral fins bud appears at 2 dph, and pectoral and pelvic fins mature at 26 dph. The pectoral and pelvic fins are still bilaterally symmetric by that stage. Then we cloned partial CDS of Tbx5 gene, which encoded a polypeptide with conserved T-domain, sharing 93% identity with zebrafish Tbx5. Phylogentic analysis reveals that this partial CDS is grouped with teleost branch, but separated with tetrapod branch. The whole-mount in situ hybridization shows that Tbx5 gene is expressed in pectoral fins, eyes, vertebra, and allantoic vesicle at 26 dph larvae of Paralichthys olivaceus. In addition, the expression of Tbx5 gene in eyes and pectoral fins are asymmetry between the left and the right sides, which is higher expressed in the eyes and pectoral fins of the left side (ocular side) than the right side (blind side). All these results suggest that Tbx5 genes may involve in the metamorphosis process of Paralichthys olivaceus.