Prokaryotic expression vector of auxin-binding protein AtTIR1 and IAA28 were constructed by gene cloning based on pGEX–KG. The expression induction conditions of two kinds of different expression strains after transfected with the recombined expression vectors were investigated. The results showed that the GST–IAA28 had the highest yields in Tuner under 0.4 mmol/L IPTG and the highest yields in BL21(DE3) at 0.6 mmol/L IPTG, with 16 ℃ the suitable inducing temperature, while the protein yields were low in Rosetta under every concentration of IPTG. GST–AtTIR1 expressed highly in Rosetta at the optimal induced condition of 0.4 mmol/L IPTG and 25 ℃, and about 0.2 mg GST–AtTIR1 was produced in 1g bacteria. But GST–AtTIR1 expressed lowly in BL21. Meanwhile, relatively purified GST–AtTIR1 and GST–IAA28 had been obtained through GST SefiroseTM resin.