Reservoirs after chemical flooding usually have residual chemicals, which can affect the driving effect of subsequent microbial drives. Among them, the effect of surfactants on the metabolites of oil-recovering bacteria is the most obvious. Therefore, this paper investigates the influence mechanism of sodium dodecyl sulfate (SDS) on the nature and structure of Extracellular Polymeric Substances (EPS) produced by metabolism of Enterobacter cloacae, through a variety of characterization to analysis the components and structure of EPS under SDS stress. The results showed that Enterobacter cloacae was identified as a glycolipid-producing strain, the main components of EPS were polysaccharides and proteins. The polysaccharide composition (%: w/w) was glucosamine, 37.2; glucose, 31.5; rhamnose, 26.3; xylose, 1.7; and unidentified sugar, 3.3; and the main component of proteins was polyglutamic acid. EPS under the stress of SDS showed an increase in the content of functional groups such as -C=O and -COOH and an increase in the cellular particle size, and production of EPS increased by 10.69 × 10³ mg/L when the SDS concentration was 2.5 × 10² mg/L; 3D-EEM results showed that the components of all three types of EPS The 3D-EEM results showed that all three types of EPS fractions contained tryptophan and protein-like substances, humic acid-like substances were only distributed in the solubilized extracellular polymers (SL-EPS), and aromatic proteins were only present in the loosely bound type (LB-EPS) and tightly bound type (TB-EPS). In addition, the peaks representing humic-like substances showed a blue shift, indicating that SDS had the greatest effect on SL-EPS. This study provides a guidance for refining the mechanism of strain EPS response to reservoir residual surfactant SDS, and provides a more comprehensive and in-depth understanding of surfactant-protein interactions.