Eukaryotic β-actin gene plays an important role in physiological process and its sequence is highly conservative as a housekeeping gene. The partial cDNA encoding β-actin in Oreochromis aureus was isolated using RT-PCR. The cDNA isolated was 424 bp encoding 138 amino acids with a calculated molecular weight of 15.5 ku. The comparison analysis of the deduced amino acid sequence of β-actin in O.aureus showed high similarity of 99.3% with those of Pagrus major, Danio rerio, Oryzias latipes, Rivulus marmoratus, and 97.8%-98.6% homology with those of other fishes, such as Carassius auratus, Takifugu rubripes. The corresponding DNA sequence of O.aureus β-actin in size of 619 bp was also cloned. By comparing the sequences of cDNA and DNA, it was found that β-actin gene in O.aureus consisted of two introns. The present study provided some useful information in designing the β-actin primers for real time RT-PCR and evaluating the β-actin expression level for different tissues.