Release of phosphorus (P) from sediment is a major source of P in many freshwater lakes. Currently, assessing the ability of sediment to release P, which is valuable to the management of water eutrophication, remains a challenge. Thus, the purpose of this study was to find effective indexes for predicting the release potential of internal-P. In this study, high-resolution diffusive gradients in thin films (DGT) and conventional sequential extraction were used to characterize the distribution and speciation of P, iron (Fe), and sulfur (S) in the surface sediment of a mildly eutrophic reservoir in southwestern China. Sediment samples exhibited large variations in Fe, S, and P, thereby providing favorable conditions for investigating the effects of Fe and S on sediment P mobilization. In contrast to traditional knowledge, our results show that total P (TP) and redox-sensitive P(BD-P) are poorly correlated with releasable P(DGT-P). This implies that high levels of sedimentary TP and BD-P do not necessarily result in an elevated release of internal-P under anaerobic conditions. Sedimentary P release was greatly suppressed at ratios of Fe/P > 30 and Fe/S > 6. Significant positive correlations between DGT-P and DGT-Fe or DGT-S suggest that Fe and S play an important role in governing the mobility of sedimentary P. These results support the combined Fe/P and Fe/S ratios as an effective and practicable index for assessing the ability of sediment to release P. Thus, our study provides a new and simple method for assessing sedimentary P pollution in freshwater ecosystems.