Midkine (MDK) and Pleiotrophin (PTN) belong to a class of heparin-binding growth factors and are highly expressed in a number of cancers. Bioactive and recombinant MDK and PTN are critical reagent for cancer drug discovery studies. MDK and PTN belong to a newly evolving family of secreted neurotrophic and developmentally regulated heparin-binding molecules. PTN is related to MDK with 45% sequence identity and both proteins have been shown to be involved in promoting neurite outgrowth. MDK is a cysteine-rich 13kDa protein containing five disulfide bonds and PTN is 19kDa protein containing ten disulphide bonds. In this study, we expressed recombinant human MDK (rhMDK), mouse MDK (rmMDK) and human pleiotrophin (rhPTN) in Escherichia coli BL21(DE3)pLysS strain. Soluble rhMDK, rmMDK and rhPTN were expressed at a high-level in this strain and the protein was purified (∼90%) by a one-step purification using heparin affinity chromatography. A total of 4mg purified MDK and 7mg of purified PTN were obtained with the overall yield from 1L of bacterial culture. Activity of purified rhMDK and rhPTN was confirmed by a cell proliferation assay using NIH3T3 cells.