The aim of this study was to clone the Actin gene from the leaf of Oncidium ‘Sweet Sugar’. Degenerate primers were designed based on the registered sequences of the Actin genes from other plants. Two Actin sequence fragments were obtained by RT-PCR. The sequencing result revealed that the two Actin gene fragments from Oncidium ‘Sweet Sugar’ contain 1062 bp with 90.87% homology, which were named as OnACT1 and OnACT2 and registered into GenBank (respective accession number: JN981136 and JN981137). They encode the same protein of 354 amino acids with three Actin signatures. Phylogenetic tree analysis indicated that Actin from Oncidium‘Sweet Sugar was closely related to the Actin of Sedirea japonica and Cymbidium faberi. The expression of Actin gene was analyzed using qRT-PCR and semi-quantitative RT-PCR. The result showed that the expression of Actin was relatively stable in different tissues at the vegetative and reproductive stage; therefore, it could be used as reference genes of the development related genes.