African swine fever (ASF) is a disease that causes severe economic losses to the global porcine industry. As no vaccine or drug has been discovered for the prevention and control of ASF virus (ASFV), accurate diagnosis and timely eradication of infected animals are the primary measures, which necessitate accurate and effective detection methods. In this study, the truncated ASFV I329L (amino acids 70–237), was induced using IPTG and expressed inEscherichia colicells. The highly antigenic viral protein I329L was used to develop an indirect enzyme-linked immunosorbent assay (iELISA), named I329L-ELISA, which cut-off value was 0.384. I329L-ELISA was used to detect 186 clinical pig serum samples, and the coincidence rate between the indirect ELISA developed here and the commercial kit was 96.77%. No cross-reactivity was observed with CSFV, PRRSV, PCV2, or PRV antibody-positive pig sera, indicating good specificity. Both intra- assay and inter-assay coefficients were below 10%, and the detection sensitivity of the iELISA reached 1:3200. In this study, an iELISA for ASFV antibody detection was developed based on the truncated ASFV I329L protein. Overall, the I329L-ELISA is a user-friendly detection tool that is suitable for ASFV antibody detection and epidemiological surveillance.