Abstract: The effects of several key factors on measuring Fe3+ reductase activity in Roots of dicotyledonous plants using 2,2'-bipyridyl-colorlmetric method were studied in nutrient solution culture under the controlled conditions. The results showed that the highest peak of absorption spectrum was 253. 3nm, the appropriate reaction time of roots in reactive solution was 2 hours. In comparison with darkness the Fe3+ reductase activity was increased significantly under light during the reaction. But aeration during the reaction (2hr.) and either immediate or still stay of reactive solution for different times (0,3.6,16hr.) after the reaction until colorimetric analysis didn't irifluence the final measuring results.