To predict B cell epitope of Ewing's sarcoma EWS/FLI-l fusion protein and to analyze its antigenicity and immunogenicity. Comprehensive algorithms were applied to predict the possible B cell epitopes of EWS/FLI-l fusion protein. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis were performed to identify the synthesized epitope peptides, ELISA assays and Western blot to detect the antigenicity, and the immunogenicity of epitope peptides. Three B cell epitopes were screened out, and HPLC and MS analysis confirmed all three synthesized epitope peptides were demandable. ELISA assays verified all three epitope peptides could prime intense antigen-antibody reaction and induce ideal antibody titers after immunization to the New Zealand white rabbit. However, Western blot confirmed that antiserum of one of these epitope peptides could not recognize EWS/FLI-1 protein. Two B cell epitopes, PQDGNKPTETSQPQ and DPDEVARRWGQRKS, derived from EWS/FLI-l protein, are identified to have potential antigenicity and immunogenicity.