Cell-free extracts of Alcaligenes sp. CKCr-6A that was isolated from Sabzevar of Iran catalyzes the NADH-dependent reduction of selenate and selenite to elemental selenium. Selenate reductase enzyme was purified with following purification process involved (NH4)2SO4 precipitation, followed by using Q-Sepharose and phenyl-Sepharose chromatography. Using gel filtration, the relative molecular mass of the enzyme was measured to be 197kDa. The outcomes showed that selenate reductase exists as a tetramer with molecular masses 70kDa, 45kDa, 35kDa and 27kDa. The Vmax and Km values of selenate reductase were 3.96μmolmin−1mg−1 of protein and 1.8mM, respectively. The Vmax and Km were detected 1.26μmolmin−1mg−1 of protein and 5.47mM towards selenite as a substrate, respectively. The enzyme revealed its optimum activity at pH6 and 35°C. Divalent cations, such as Ca2+ and Mg2+, had no effect on the activity, while similar concentrations of Pb2+and Cu2+ and some Metabolic poisons such as N-ethyl maleimide eliminated the activity. In addition analysis discovered the role of Mo+2 in selenate reductase. Results showed that [EMIm][Br] have a positive effect on activity and thermal stability of the reductase and also is effective on the amount and size of synthesized Se-NPs that mediated by this enzyme.