Abstract: Orthogonal test was applied to quantitative analysis of Si, Mn, Cr and Ni four elements content of 304L stainless steel standard sample by SEM and energy dispersive spectroscopy(EDS), and the significance of different test factors such as accelerating voltage, live time, spotsize and working distance on the measured results was analyzed, and the best test conditions were obtained by range analysis and variance analysis. The result show that the most significant factor was accelerating voltage, the significance of live time and working distance was quite similar, and the minimal significant factor was spotsize. The optimal test conditions were as following with accelerating voltage being 20 kV, live time being 100 s, spotsize being 5.0 μm, working distance being 10 mm. The measured four elements content under the optimal conditions were all very close to the standard values.