Objective Four B and Th cell epitopas were selected from conservative domain of Plasmodium falciparum antigens to construct two groups of chimeric malaria DNA vaccines with different configurations and their antigenicitles were studied. Methods The partially synthesized oligonucleotide was annealed, PCR amplified and cloned into a mammalian cell expression vector. By using a pair of isocaudamers on the vector, different single copies of B epitopes were multiplied and were tenderly stdnged into two groups of chimeric DNA vaccine with different configurations, BALB/c mice were immunized with these DNA plasmids by either intramuscular or intraderrnal injections. Results The antisera from the immunized mice tested by ELISA showed that only the configuration which had a single copy of universal T helper cell epitope, CS. T3, located at the C terminal of the multi-copy B ceil epitopes induced a high antibody response. The T helper cell epitope at any other position of the peptide, or the double T helper cell epitopes configured with the B cell epitopes did not enhance antibody response, and some configurations even decreased the hurnoral response to a B cell epitope. Conclusion This study demonstrated that both combination and configuration of the epitope may affect the antigenicity of a chimeric multiple antigen.