Cytokeratin 18 (K18) is an ‘S’ keratin and is the first of the intermediate filament proteins to be expressed during mammalian and amphibian development. According to the published conserved DNA sequences of keratin 18 from human, xenopus and zebrafish ,two pairs of primers were designed to obtain 2 779 bp of keratin 18 gene derived from the genome DNA of Gobiocypris rarus by PCR. And then through the genome walking upstream and downstream sequences were also amplified. And then the full amplified fragment of keratin 18 was inserted into Escherichia coli cloning vector pMD19T and sequenced. The result indicated that the keratin 18 of Gobiocypris rarus was successfully obtained about 3 765 bp. The analysis of NCBI's blastn showed that comparing Gobiocypris rarus with zebrafish ,the keratin 18 DNA sequence was highly conserved with 73.6% similarity. Finally, biologically analysis and ORF prediction showed that the keratin 18 DNA sequence contained 7 exons and 6 introns, and coded for a protein with 367 amino acids.