Objective To prepare and characterize the monoclonal antibodies (mAbs) against chicken interleuikin-4 (ChIL-4). Methods Using lymphocyte hybridoma technique, 8-week-old BALB/c mice were immunized with purified recombinant ChIL-4 (rChIL-4) with GST tag (GST-ChIL-4), and rChIL-4 with histidine tag (His-ChIL-4) was utilized to set up an indirect ELISA to screen positive hybridomas. Limited dilution methods were used to generate hybridoma cell lines stably secreting mAbs against ChIL-4. The specificity of the mAbs was identified by indirect ELISA, immunofluorescent assay (IFA) and Western blot. Results Four mAbs against rChIL-4 were obtained and named 6A8, 18F12, 19F1 and 20G3. ELISA titers of the 4 mAbs were (1.6~6.4)×104. ELISA, IFA and Western blot showed that the 4 mAbs could only react with rChIL-4, but not with the other proteins like bovine IL-4 (BovIL-4), chicken IFN-γ (ChIFN-γ). Conclusion The mAbs against rChIL-4 were successfully obtained.