The ω-atracotoxin-Ar1b toxin (ω-ACTX-Ar1b) is one of the arthropod-selective peptide neurotoxins from the venom of Australian funnel-web spider Atrax robustus. The gene of Ar1b was synthesized and cloned into pET-32a(+) vector to allow expression of Ar1b as a fusion protein with thioredoxin and the His-tag (rTrx-Ar1b) in E. coli BL21 (DE3). The optimal condition for inducing the expression of rTrx-Ar1b was 1.0 mmol L-1 IPTG for 6 h at 28°C. The fusion protein rTrx-Ar1b was expressed in soluble form and was purified effectively by HisTrap HP affinity column and rpHLPC and a final yield of purified rTrx-Ar1b was 95 mg from 1 000 mL E. coli culture. The LD50 values for Mythimna separate and Tenebrio molitor were 111.66 and 11.04 μg g-1 determined by injection of the purified rTrx-Ar1b. The results indicated that the recombinant Ar1b protein was successfully expressed in E. coli and it was high toxicity against tested insects. The ω-atracotoxin-Ar1b toxin (ω-ACTX-Ar1b) is one of the arthropod-selective peptide neurotoxins from the venom of Australian funnel-web spider Atrax robustus. The gene of Ar1b was synthesized and cloned into pET-32a vector to allow expression of Ar1b as a fusion protein with thioredoxin and the His-tag (rTrx-Ar1b) in E. coli BL21 (DE3). The optimal condition for inducing the expression of rTrx-Ar1b was 1.0 mmol L-1 IPTG for 6 h at 28 ° C. The fusion protein rTrx-Ar1b was expressed in a soluble form and was purified effectively by HisTrap HP affinity column and rpHLPC and a final yield of purified rTrx-Ar1b was 95 mg from 1 000 mL E. coli culture. The LD50 values ​​for Mythimna separate and Tenebrio molitor were 111.66 and 11.04 μg g-1 determined by injection of the purified rTrx-Ar1b. The results indicated that the recombinant Ar1b protein was successfully expressed in E. coli and it was high toxicity against tested insects .