[Objective] We cloned and expressed the agnE gene involved in nicotine-degrading in Agrobacterium tumefaciens and characterized the AgnE protein. [Methods] The agnE gene fragment (1029 bp) was amplified with PCR. Recombinant plasmid pET28a-agnE was constructed and transformed into Escherichia coli BL21(DE3) for heterologous expression, and the expressed protein was detected with SDS-PAGE. The enzyme activity of AgnE was determined spectrophotometrically by monitoring the decrease of 2,5-dihydroxy pyridine at 320 nm. The effect of temperature, pH and metal ion on enzyme activity was investigated. [Results] The agnE gene was cloned and the recombinant plasmid pET28a-agnE was expressed. The molecular weight of the recombinant protein was 42.0 kDa, and the protein was expressed in the form of inclusion bodies in cells. The AgnE protein had high enzyme activity at pH 8.0 and and 20 ℃. In addition, Fe²⁺ showed significant promoting effect on enzyme activity. [Conclusion] The activity of AgnE protein with 2,5-dihydroxy pyridine dioxygenase was clarified.