〔Abstract〕 Objective To humanize the variable region of the IL9-2G1 rabbit monoclonal antibody (IL9 2G1 mAb) and to evaluate its biological activity. Methods The humanized sequences of IL9-2G1 mAb were designed. The heavy chain variable region (VH) and light chain variable region (VL) of the antibody were humanized and cloned into vector backbones containing the constant region frameworks of human IgG1 and Ig Kappa subtypes. Protein purification was carried out using the Chinese hamster ovary cell (CHO) expression system. Enzyme-linked immunosorbent assay (ELISA) was used to qualitatively analyze the antigen-binding activity ofthe antibodies. After screening the optimal humanized antibody candidate, surface plasmon resonance (SPR) was performed to detect molecular affinity. Western blot was used to examine the effects of the rIL-9 group and the rIL-9 + hIL-9 mAb group on the phosphorylation of Janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3), while ELISA was employed to assess the impact of the Th9 group and the Th9 + hIL-9 mAb group on interleukin-9 (IL-9) secretion. Results Purification and ELISA results showed that the heavy chain OH and light chain FL exhibited good compatibility and expression performance. SPR results indicated that the equilibrium dissociation constant (KD) of M501042-OHFL against the analyte M30903 was 1.33×10_7 M, which was superior to the parental 2G1 antibody. The Western blot results showed that the levels of phosphorylated JAK1 (p-JAK1) and phosphorylated STAT3 (p-STAT3) were significantly decreased in the rIL-9 + hIL-9 mAb group compared with the rIL-9 group (P<0.01). The ELISA results indicated that the secretion of IL-9 was significantly reduced in the Th9 + hIL-9 mAb group compared with the Th9 group (P<0.01). Conclusion Humanization modification is performed on the variable region of IL9-2G1 rabbit monoclonal antibody, and the humanized antibody hIL-9 mAb OHFL with high affinity and strong binding stability is successfully constructed, which provides an experimental basis for subsequent research.