Abstract: This study aimed to clone the protein coding sequence（CDS） of retinoic acid-related orphan receptor alpha（RORα） in Bos taurus. The structural and functional characteristics of the gene were predicted and analyzed, the overexpression effect of the eukaryotic expression vector in HEK293T cells was verified, and the expression profile of the gene in different tissues of dairy cows was further detected. In this study, total RNA from liver tissue of Bos taurus and cDNA was obtained by reverse transcription. After PCR amplification, the CDS region fragment of Bos taurus RORα gene was obtained. It was ligated with the linearized vector pcDNA3.1-Puro-N-3HA by homologous recombination. After transforming competent cells DH5α, the recombinant plasmids initially identified as positive clones were sequenced. Combined with the sequencing results, bioinformatics softwares such as ExPASy, Protscale and DNAstar were used to predict and analyze the physicochemical properties and functional properties of Bos taurus RORα protein. Then, the control group empty plasmid pcDNA3.1-Puro-N-3HA and the experimental group recombinant plasmid pcDNA-3.1-3HA-RORα were respectively transfected to HEK293T cells, the overexpression effect of Bos taurus RORα gene was detected by real time quantitative PCR and western blotting techniques. Finally, the relative expression of Bos taurus RORα gene in seven tissues including liver, spleen, and muscle tissue was detected by semi-quantitative RT-PCR technology. The PCR results showed that the CDS region fragment of the Bos taurus RORα gene was successfully cloned. The results of enzyme digestion and sequencing showed that the recombinant plasmid pcDNA3.1-3HA-RORα was successfully constructed. The results of bioinformatics software prediction analysis showed that the tertiary structure of Bos taurus RORα protein was highly similar to that of Capra hircus and Mus musculus. The comparison of homology among different species showed that Bos taurus RORα gene was highly similar to that of Capra hircus. The results of real time quantitative PCR and western blotting showed that compared with the control group, the mRNA and protein expression levels of Bos taurus RORα gene in HEK293T cells in the experimental group were significantly increased. The results of semi-quantitative RT-PCR showed that the expression level of Bos taurus RORα gene was the highest in liver and the lowest in spleen among the seven tissues tested. In this study, the CDS region of Bos taurus circadian clock gene RORα was successfully cloned, and the expression effect of its eukaryotic expression vector at mRNA and protein levels was verified in HEK293T cells. The structural and functional characteristics of Bos taurus RORα gene were analyzed, and its expression profile in different tissues was detected. This study provided a preliminary basis for further exploring its mechanism of action in Bos taurus reproductive and metabolic regulation.