Microbial enhanced oil recovery (MEOR) technology is an environmental-friendly EOR method that utilizes the microorganisms and their metabolites to recover the crude oil from reservoirs. This study aims to research the potential application of strain SL in low permeability reservoirs. Strain SL is identified as Bacillus subtilis by molecular methods. Based on the mass spectrometry, the biosurfactant produced by strain SL is characterized as lipopeptide, and the molecular weight of surfactin is 1044, 1058, 1072, 1084 Da. Strain SL produces 1320 mg/L of biosurfactant with sucrose as the sole carbon source after 72 h. With the production of biosurfactant, the surface tension of cell-free broth considerably decreases to 25.65 ± 0.64 mN/m and the interfacial tension against crude oil reaches 0.95 ± 0.22 mN/m. The biosurfactant exhibits excellent emulsification with crude oil, kerosene, octane and hexadecane. In addition, the biosurfactant possesses splendid surface activity at pH 5.0–12.0 and NaCl concentration of 10.0% (w/v), even at high temperature of 120 °C. The fermentation solution of strain SL is applied in core flooding experiments under reservoir conditions and obtains additional 5.66% of crude oil. Hence, the presented strain has tremendous potential for enhancing the oil recovery from low-permeability reservoirs.