Biosurfactants produced by native microorganisms have excellent surface activity and ideal environmental compatibility, and the mining of the biosurfactant-producing strains has become a key focus in the field of applied and environmental biotechnology. In this paper, we report a new lipopeptide-producing strain isolated from the production water of Daqing oilfield in China and identified as Bacillus Subtilis R1-2 based on 16S rRNA gene sequence analyses. A combination of ESI-MS and FT-IR analyses revealed that the strain R1-2 produced the surfactin family containing four members of the C₁₂-surfactin, C₁₃-surfactin, C₁₄-surfactin and C₁₅-surfactin, which is a representative family of the lipopeptide biosurfactants. The lipopeptide biosurfactant produced by the strain R1-2 exhibits excellent surface activity and good thermal stability over a temperature range between 20 °C and 100 °C and pH range between 3 and 14, and has a strong salt tolerance to NaCl concentration up to 140 g/L. In addition, the lipopeptide biosurfactant demonstrates significant properties in changing the contact angles of oil reservoir core slices from 86.2° to 39.0° and the wettability from strong oil-wet to strong water-wet, and therefore, resulted in a good oil removing ability with an efficiency of 64.84%, suggesting that the lipopeptide-producing strain R1-2 is promising in applications in environmental bioremediation and enhanced oil recovery.