To explore the change trend of intermediate products in biotransformation of 2, 4-dinitrotoluene (2, 4-DNT) and properties of nitroreductase produced by Rhodobacter sphaeroides, the effect of different combination of nitrogen sources on the efficiency of biotransformation 2, 4-DNT by Rhodobacter sphaeroides and the growth of Rhodobacter sphaeroides cells was studied. The concentration changes of 2, 4-DNT and biotransformation products of 2, 4-DNT were measured at different time. The patterns of isoenzymes of nitroreductase was analyzed using native-polyacrylamide gel elecrophoresis (native-PAGE) method, and kinetic parameters of the nitroreductase were measured and obtained. Results show that the optimum combination of nitrogen sources to biotransformation of 2, 4-DNT by Rhodobacter sphaeroides is complex nitrogen sources composed of yeast extract and (NH₄)₂SO₄. Using Rhodobacter sphaeroides, 2, 4-DNT can efficiently reduced as 4-amino-2-nitrotoluene(4A2NT) and 2-amino-4-nitrotoluene(2A4NT). However, the content of 4A2NT is far greater than that of 2A4NT, they have not been futher reduced to 2, 4-diaminotoluene (2, 4-DAT). When the initial concentration of 2, 4-DNT is 20, 40 and 60 mg·L^-1, there is an obvious promoting effect on the actiyity of nitroreductas produced by Rhodobacter sphaeroides. While, when the initial concentration of 2, 4-DNT is 80 mg·L^-1, the actiyity of nitroreductase is inhibited. The activity of nitroreductas produced by Rhodobacter sphaeroides is the highest at the initial pH=7. The kinetic parameters, the maximum reaction rate V_max and Michaelis constant K_m, for the nitroreductase in Rhodobacter sphaeroides are 0.0507 mmol·(L·min) ^-1 and 0.1772 mmol·L^-1, respectively.