Real-time fluorescence quantitative PCR is an effective method to quantify the transcriptional profile of target genes. Use of proximal gene as internal reference is essential when performing qRT-PCR experiments. The Actin (ACT) gene is highly conserved cross species and expressed stably and is often used as an internal control. In order to obtain the ACT gene of cauliflower, the ACT gene of cauliflower was cloned by RT-PCR method (GenBank ID: MG598643)1. The open reading frame (ORF) is 1,134 bp, encoding 377 amino acids with a predicted molecular weight of 41.77 kD and a hypothetical isoelectric point of 5.395. Wolf Psort analysis indicated that BobActin protein was located in the cytoplasmic matrix, and Motif Scan analysis showed that BobActin protein had the conserved actin at position of 4-377 sites. BobActin shared 90% identity with the homologous proteins from genus Brassicacea, such as Brassica oleracea var. oleracea, Brassica rapa and Brassica napus. A pair of qRT-PCR primers was designed from the BobActin gene sequence, and this combination showed high specificity and amplification efficiency. qRT-PCR analysis indicated that the BobActin gene was stably expressed in tissues of cauliflower, including root, stem, flower ball, leaf, even under various stress treatments (low temperature, high temperature, salt, drought and ABA). Thus, this gene might serve as an internal reference being suitable for gene expression in cauliflower.