In this paper a novel method for determination of Fe in uranium matrix has been demonstrated using energy dispersive X-ray fluorescence (EDXRF) technique. Effects of various parameters such as variation of matrix concentration, detection limit, accuracy, precision, comparison with other established methods etc. were investigated in detail. Analysis of synthetic standards as well as real samples shows that for ten determinations relative standard deviation (RSD) is <2 %. The method can be used for determination of 2.24–3500μgmL^-1 Fe in uranium matrix with high accuracy and precision.