The fluorescence effect induced by Kβ photons is usually so small that it can be neglected. However, in the Fe–Mn system, omitting Kβ fluorescence correction will lead to the overestimation of the Mn content especially when Mn is the minor alloy element. In this study, the error in the Mn concentration induced by Kβ fluorescence was investigated by both Monte Carlo simulation, using the pyPENELOPE program, and systematic electron probe measurements on the Fe–0.53% Mn alloy standard by the aid of CalcZAF software. It is shown that the error caused by Kβ fluorescence exceeds 4% for the Fe–0.53% Mn alloy. The problem can be overcome by utilizing CalcZAF in which β-line fluorescence has been included, or by employing a similar standard Fe–0.85% Mn for Mn in the absence of β-line fluorescence correction. In addition, a modified calibration curve method, using k-values instead of X-ray intensity as a variable, is presented and used to measure the Mn concentration. The accuracy of this method is as good as or better than that of the conventional matrix correction method. Compared with conventional calibration curve methods, it is time-saving because the k-value is not sensitive to instrument fluctuations and the established curve remains valid for a long period.