Trace iron (Fe) has been found to be critical for improving the catalytic activity of heteroatom-doped carbon materials for oxygen reduction reaction (ORR). However, the exact roles played by the trace Fe in the catalysts have not been studied in detail. In this study, nitrogen (N) and sulfur (S) dual-doped carbon nanospheres were synthesized with and without trace Fe addition, named NSFeDC and NSDC, respectively. NSFeDC showed obviously stronger catalytic activity for ORR than NSDC. The microstructures of these two catalysts were characterized comprehensively. After adding the trace Fe, the contents of N and S in NSFeDC were 8.1 and 3.3 times higher than those in NSDC. The functional group composition analysis demonstrated that one part of the increased N/S formed the chemical bond of Fe-N and Fe-S, and the other part was involved in the abundant extra formation of metal-free N/S-based active site, such as pyridinic N and -C-S-C-. The underlying mechanism is that the trace iron not only participated itself in constructing the active sites, but also served as the catalyst for the formation of N/S-based active sites. The comparison between the TEM images of NSDC and NSFeDC clearly showed that the size of the nanosphere increased after adding the trace Fe. This was probably because the iron atom connected two separate carbon groups together by bonding with the nitrogen atoms. Besides the enlarged particle size, the surface area of NSFeDC decreased by nearly one half compared to that of NSDC.