Inconel 718 (IN718) nickel-based superalloy is widely used in aerospace and nuclear applications owing to its excellent comprehensive mechanical properties, oxidation resistance, and hot corrosion resistance. However, the elemental segregation caused by heterogeneous solidification during casting has great influence on the mechanical properties. Therefore, accurately characterizing the segregation behavior is necessary. Traditional quantitative characterization of elemental segregation uses various sampling methods, in which only macroscopic segregation results are obtained. In this study, micro-beam X-ray fluorescence (μ-XRF) is used for the quantitative characterization of element micro-segregation in IN718 superalloy. The concentration distributions of Cr, Fe, Mo, Nb, and Ti in IN718 alloy are determined with optimized testing parameters, and the degree of elemental segregation in different regions of the analytical area is calculated. It is found that the segregation degree of Nb and Ti in the testing area is larger than other alloying elements. The correlation between the microstructure distribution and the segregation degree of Nb and Ti has been studied using scanning electron microscopy (SEM) combined with energy-dispersive spectrometry (EDS). There is severe segregation of Nb and Ti in areas where Nb-containing precipitates are accumulated. The distribution of abnormal signals of Nb with a high fluorescence intensity has a close relationship with the area of precipitates-enriched Nb.