Protein kinase CK2 is a heterotetramer ser/thr protein kinase composed by two catalytic subunits (α or α′) and two regulatory subunits (β). The recombinant plasmid containing human CK2β subunit cDNA was transformed into E.coli BL21(DE3) and expressed induced by IPTG. Most of the expressed CK2β proteins were insoluble. From 6 L(about 10.4 g) bacteria, 20 mg soluble protein was extracted from the insoluble pellet and purified by a single step P11 phosphocellulose chromatography, the yield of purified CK2β subunit was 6.8 mg. SDS-PAGE analysis of the purified protein showed only one band with molecular mass of 26 ku. Western blot analysis confirmed that the expressed product was human CK2β subunit. Addtion of the CK2β subunit to CK2α subunit led to maximum stimulation at a 1∶1 molar ratio of both subunits. These results demonstrated strongly that the cloned, expressed and purified recombinant protein was human CK2β subunit. The large amount of purified recombinant CK2β protein lays solid basis for further study directly the characteristics of the enzyme and the relationship of structure and function between CK2β subunit and its interacted proteins.