Tessier sequential extraction procedure was employed to analyze the heavy metal speciation of river sediment. Comparisons were made of the biological availability method, leaching characteristics method, geo-accumulation index method, and potential ecological risk method of heavy metals before and after washing by bio-surfactant sophorolipid. Dissolved organic matter in sediment was also evaluated by a three dimensional fluorescence analysis. Results showed that washing by bio-surfactant greatly changed the speciation of heavy metals in the sediment. Heavy metals became less migratory thus resulting in decreased bioavailability and decreased hazard. Washing also led to less leaching and lower potential individual or comprehensive risk to eco-environment. Our results revealed that washing by sophorolipid was effective in reducing the hazardous effect of heavy metal to environment. After washing, heavy metals were more stable and presented lower risk thus washed sediment could be considered for reuse.