Abstract: Nanoparticles (NPs) are widely used in the petroleum industry, particularly in upstream and downstream applications, due to their quantum effects and high surface area-to-volume ratio. However, stable dispersion and high input ratios of NPs are critical factors limiting their application. This study thus synthesizes the biopolymer nanocomposite E-Ag NPs in one step using the metabolic product exopolysaccharide (EPS) from Bacillus subtilis as both a reducing and stabilizing agent. This study explored the potential of E-Ag NPs to enhance oil recovery in low- and medium-permeability formations. Experimental results showed that the average particle size of Ag NPs synthesized using EPS ranged from 20–50 nm. The presence of EPS resulted in a higher Zeta potential value (−43.4 mV), indicating improved stability of the nanofluid, effectively inhibiting NPs aggregation. Interfacial tension and wettability evaluation experiments demonstrated that E-Ag NPs exhibited excellent capabilities in reducing interfacial tension and altering wettability. In the enhanced oil recovery evaluation experiments, the imbibition experiment with E-Ag NPs achieved a maximum recovery rate of 54.32%, and the core flooding experiment reached a maximum recovery rate of 16.33%. In conclusion, the biopolymer nanocomposites developed by this method offer valuable guidance and reference for oilfield development, providing a green and efficient potential solution for enhanced oil recovery.