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The preparation of anti-idiotypic antibodies is an innovative strategy for developing novel insecticidal protein resources. To design and construct insecticidal protein resources by engineering antibody technology, two animalized single-chain variable fragments (scFv) against Plutella xylostella larvae were constructed by CDR grafting. The amino acid sequences of framework regions (FRs) in the heavy chain variable (VH) and light chain variable (VL) of the swine and bovine antibodies were obtained from the NCBI database. Subsequently, the complementarity determining regions (CDRs) of the humanized anti-idiotypic scFv 3E1 were grafted onto the FRs of the swine and bovine antibodies, respectively. The reconstructed sequence was analyzed and synthesized, and the phage displaying the pIT2 plasmid was acquired by substituting the pUC57 vector. Moreover, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was employed to typify and characterize the CDR grafting scFv. The binding interaction between the animalized scFv and the brush border membrane vesicles (BBMV) of P. xylostella was analyzed using surface plasmon resonance (SPR) analysis. Furthermore, the biological activity of the animalized scFv with grafted CDRs against P. xylostella was determined by the leaf dipping assay. The results showed that both the swine-3E1-pIT2 and bovine-3E1-pIT2 plasmids were obtained by replacing the original vector PUC57. Furthermore, both the swine-pIT2 and bovine-pIT2 exhibited 44.3% and 43.0% inhibition of the bindings of Cry1B to its polyclonal antibody, respectively. The results indicated that both the swine-pIT2 and bovine-pIT2 could be classified as β subtype anti-idiotypic scFvs, which could simulate the partial structural features of Cry1B. Subsequently, the binding affinities of swine-pIT2 and bovine-pIT2 with P. xylostella BBMV were determined by SPR analysis to be 209.48 and 195.31 RU, respectively. The bioassay results indicated that swine-pIT2 and bovine-pIT2 exhibited insecticidal activity against P. xylostella larvae. Both LD50 values of the swine-pIT2 and bovine-pIT2 were 5.90 × 107 and 6.22 × 107 CFU/mL, respectively, which were lower than the LD50 of 3E1 (6.86 × 107 CFU/mL). Moreover, the toxicity regression equations for the two modified materials had slopes of 1.50 and 1.48, respectively, surpassing the slope of 3E1(1.23). In summary, both swine-pIT2 and bovine-pIT2 are classified as the β subtype anti-idiotypic scFvs, which exhibit the ability to bind to P. xylostella BBMV and demonstrate insecticidal activity. This innovative approach provides a new method for exploring novel biopesticide protein resources. [ABSTRACT FROM AUTHOR]