Grouper (Epinephelus coioides) is one of the important farmed fish in the southern Taiwan. However, grouper aquaculture in Taiwan has a serious problem of infection, especially in grouper larvae breeding stage. The infection resulted in very high mortality, which causes massive economic loss. Therefore, early detecting the presence of pathogen is critical for preventing epidemic outbreak. Interleukin-1 (IL-1) is one of proinflammatory cytokines that form a feedback control loop with anti-inflammatory cytokines to maintain the homeostasis of host immune response. The increase of IL-1 expression could be an indicator of pathogenic insult. In this study, total RNA of Epinephelus coioides fertilized egg was extracted for reverse transcription-polymerase chain reaction (RT-PCR) to amplify cDNA of IL -1£]. The cDNA amplified was then cloned into pGEX4T-3 for the expression and purification of GST-IL-1£] fusion protein. GST-IL-1£] fusion protein purified was then used to immunize New Zealand white rabbit for generation of antiserum against IL-1£]. Western blot result confirmed the specificity of antiserum as the immune serum, but not the preimmune serum, detected the immunogen GST-IL-1s. Further experiments using live Epinephelus coioides injected with or without lipopolysarcharides (LPS) further confirmed that this antiserum could detect a massive increase of IL-1£] protein after the injection of LPS in either protein lysate by western blotting or in frozen tissue section of head kidney by immunohistochemistry. In summary, we successfully generated a rabbit specific antiserum against IL-1£] of Epinephelus coioides , which could be a useful reagent for future analysis of fish immune response upon pathogen infection.