Objectives To develop a cost-effective ELISA for detection of antibodies against infectious bronchitis virus (IBV) by using a multi-fragment protein as coating antigen. Results A multi-fragment antigen, termed BE, which was composed of eight antigenic fragments selected from the three major proteins (S, M, and N) of IBV, was expressed in Escherichia coli. The entire protein had a molecular weight of 61.5 kDa. In addition to it, a smaller truncated protein was also produced; both could react with IBV-positive serum. Next, an indirect ELISA (BE-ELISA) was developed. Coefficients of variation of this assay were lower than 10 %, and no cross-reactivity between the coated antigen BE and antiserum against newcastle disease virus, avian influenza virus, or infectious bursal disease virus was observed. The performance of BE-ELISA was evaluated, and showed 95.4 % coincidence ratio with the whole virus based-ELISA (IDEXX). Conclusions The multi-fragment antigen (BE) may represent a promising alternative to the whole virus without safety problems, and this newly established ELISA provides an effective method for anti-IBV antibody detection.