Rho kinase (ROCK) inhibitors are effective candidates for treating nerve or myocardial injury, erectile dysfunction, and other cardiovascular diseases. Purified ROCK is a foundation for ROCK inhibitors screening and for its function research in vitro. This article established an easy way to harvest active recombinant ROCK catalytic domain (ROCK-CD) of rat in Escherichia coli (E. coli). The cDNA of ROCK-CD was amplified by RT-PCR, and subcloned to pET28a(+) vector to express the protein in E. coli BL(21) as inclusion bodies. The protein was purified by HiTrap chelating column, and its refolding was achieved by gradient dilution from guanidine hydrochloride solution, and desalinated by ultrafiltration. The result of DNA sequencing and protein sequence analysis indicate there were three amino acid residua of mutation, but the activity was not significantly affected. The activity of the recombinant protein was confirmed by ROCK II activity fluorescence polarization kit. Therefore, this is an easy and rapid procedure to harvest a large quantity of activity recombinant ROCK-CD at low cost.