The Huaihe River has suffered increasing pressure from pollutants including metals from anthropogenic activities. In this study, enrichment and fractionation behavior of trace metals were analyzed in sediment samples obtained from fish spawning area of the Huaihe River (Anhui Section) to evaluate the potential ecological risk of trace metals to aquatic organisms. Geochemical indices including enrichment factor and geo-accumulation index as well as mean probable effect concentration quotient and risk assessment code were adopted to assess the contamination degree and potential ecological toxicity. Results showed that the total contents of Cu, Pb, Zn, Cr, Cd, As, and Hg in sediment were 23.1 ± 6.4, 32.3 ± 11.1, 76.8 ± 14.2, 84.6 ± 17.2, 0.2 ± 0.1, 9.0 ± 3.0, and 0.031 ± 0.010 mg/kg, respectively. The indexes EF and Igeo revealed slight accumulation for Pb, Zn, Cr, Cd, and As in some sampling sites. The result of Qm-PEC demonstrated that trace metals in sediment were not toxic to aquatic organisms. Most trace metals appeared to mainly associate with the residual form suggesting lower mobility whereas Cd presented a relative higher exchangeable fraction indicating a great degree of bioavailability. The result of risk assessment code (RAC) evaluation revealed that Cd poses a medium ecological risk for aquatic organisms whereas most of the other trace metals pose low risks.