Much attention has been directed toward amphiphilic nanosheets in diverse applications owing to their remarkable properties. In this work, the rhamnolipid-molybdenum disulfide nanosheets, environmentally friendly biological-amphiphilic-nanosheets (BANs), are prepared through the one-step simple hydrothermal method. The ultralow concentration of BANs (0.005 wt %), as a solid emulsifier, can emulsify crude oil and stabilize the size of emulsions (oil in water) within 1.2 μm. The adsorption of BANs to the oil−water interface improves the stability of the BANs emulsion, and it both reduces interfacial tension and increases interfacial film strength. Tremendous desorption energy (thousands of K B T) is required to remove a single BAN from the interface to the bulk phase. Besides, BANs can turn an oil-wet surface into an intermediate-wet surface and spread to exfoliate oil film on the solid surface. The evolution of the three-phase contact line experiences two stages: a steep stage and a gentle stage. The spreading behaviors are due to an imbalance between the surface forces and structuring disjoining pressure at the confinement region (wedge film). Moreover, 25.3% of additional oil recovery was observed from the flooding of an ultralow concentration of BANs nanofluid (0.005 wt %) in a natural outcrop core, which is 2.5 mD in permeability at experimental conditions: 120 °C and around 25 × 10 4 mg/L in salinity. The high tolerance of rhamnolipid-molybdenum disulfide nanosheets to high temperature and harsh salinity is because of synergism between MoS 2 nanosheets and rhamnolipid molecules. Additional residual oil reduction is due to a combination of multiple effects such as crude oil emulsification, wettability alteration, interfacial tension reduction, and formation of structuring disjoining pressure. This study presents the possibility to enhance oil recovery in low-permeability reservoirs with harsh conditions.