A cDNA library from the brain of Andrias davidianus was constructed using the CloneMinerTM cDNA Library Construction Kit. The titer of the primary library was 1.5×105 cfu·mL^-1, and the recombined efficiency of cDNA library was 91.70%. The average insert size was 0.791 kb, while the titer of amplified library was 4.0×109 cfu·mL^-1. The thyroid-stimulating hormone β-subunit (TSHβ) cDNA was obtained through sequencing clones picked randomly, suggesting some full length cDNAs were contained in the library. TSHβ was 705 bp in length, including an open reading frame of 417 bp. The deduced TSHβ subunit contains 138 amino acids consisting of a putative signal peptide of 19 amino acides and a putative mature protein of 119 amino acides. The deduced amino acides of the Chinese giant salamander TSHβ mature protein shares identities of 53.6%, 52.2% and 60.3% separately with bullfrog, Japanese toad and xenopus. The vertebrate TSHβ protein sequences had been used to construct Neighbor-joining (NJ) and Maximum parsimony (MP) phylogenetic tree. As analyzed, Andrias davidianus derived from amphibia firstly, which could reflect its original evolutionary status. RT-PCR analysis indicated that the TSHβ mRNA was expressed abundantly not only in pituitary but also in gonads.