Vascular endothelial growth factor (VEGF), the most potent endothelial cell mitogen and also a regulator of vascular permeability, is emerging as a powerful prognostic tool. The aim of the present study is to produce specific rabbit polyclonal antibodies against bacterially expressed human VEGF165 using a recombinant DNA approach. A plasmid has been constructed to direct the synthesis of recombinant human VEGF165 (rhVEGF165) in Escherichia coli as a fusion protein containing a His6-tag at the N-terminus. The rhVEGF165 was purified based on its 6xHistag by affinity chromatography using Nickel-Agarose Column and refolded by dialysis. Two rabbits were immunized with the purified recombinant protein to produce polyclonal anti-human VEGF antibody. The titer was checked with indirect ELISA and the final titer was 1:25600.We used the purified recombinant protein and polyclonal antibody to detect VEGF expression levels in the serum of 33 tumor patients and 25 healthy controls by an established sandwich ELISA. The mean antigen concentration in serum of patients was approximately 407 pg/ml and significantly higher than those in the healthy controls in which the circulating VEGF antigens concentration was not detectable (P <0.05). Thus, this newly ELISA could be a rapid, effective and cheap method for tumor diagnosis using serum VEGF as a biomarker.