In the present study, a novel rod-shape hybrid monolithic column has been prepared by surface-initiated atom transfer radical polymerization. Compared with the traditional monoliths, the monolith could trend to form the 3-dimensional bicontinuous skeleton structure with macroporous, because of low concentration of free radical and the stepwise growth of polymer chains in atom transfer radical polymerization. Through a novel catalyst system of combining iron ferrous chloride and carbon tetrachloride, the monolith was made from Glycidyl methacrylate, Ethylene glycol dimethacrylate, dodecyl alcohol, and methyl alcohol. The monolithic column was characterized by various techniques, such as Scanning electron microscopy and Fourier transform infrared spectroscopy. The monolithic column was used for protein separation and the method reproducibility was tested by evaluating the intra- and inter-day precisions. The results show that the monolith column according to atom transfer radical polymerization exhibits good separation capability through the testing analysis.