Abstract: In this paper, batch experiments were carried out by treating simulated uraniumcontaminated groundwater with Shewanella putrefaciens, and uranium in extracellular polymeric substances (EPS) was extracted using three different methods. Moreover, speciation of uranium in EPS was investigated by filtration and inductively coupled plasma with mass spectroscopy. The sequential NaHCO3 and HNO3 extraction approach was applied to fractionate uranium in the Shewanella putrefaciens during uranium immobilization reaction was performed. The changes before and after the reaction between EPS and uranium were characterized by Fourier transform infrared spectrometer (FTIR). The results show that the cation exchange resin (CER) method was suitable for characterizing the uranium content and form in EPS, and about (14.0±1.0)% of the total uranium immobilized by Shewanella putrefaciens was found to be present in EPS. The analysis of single particle inductively coupled plasma mass spectrometry (SPICPMS) shows that the particle uranium in the EPS obtained by the CER method is the main form. Biosorption and biomineralization are confirmed to be involved in the uranium immobilization by EPS. The FTIR spectra further show that uranium may combine with the carboxyl functional group in EPS. The findings of this study imply the important roles of EPS in the immobilization of uranium in groundwater using Shewanella putrefaciens.