Abstract: MCNP simulation was utilized to study the effects of substrate materials and X-ray energy on X-ray RoHS fluorescence analysis. The linear relationship between signal intensity and concentration was well maintained for low concentration, while slopes were significantly different in presence of different substrates. The simulation results of Cd element indicate that the absorption coefficient can influence the fluorescence intensity by up to two orders of magnitude. Therefore, the absorption calibration factor of fluorescence intensity was introduced to improve the accuracy of the concentration measurement. Then, one calibration standard can be used for different substrates by overcoming variation of concentration conversion data.