Biosurfactant is a specific natural surfactant produced by the microorganism. Its adsorption on rock surface has a great impact on the change of reservoir wettability. The mechanism of wettability alteration is crucial to the oil recovery. In this paper，the interaction between rhamnolipid biosurfactant and hydrophobic glass surface was studied by contact angle experiment，oil displacement experiment，free energy of liquid-solid interface measurement and molecular dynamics simulation，explaining the adsorption effect of rhamnolipid biosurfactant on the hydrophobic glass surface’s wettability. The results showed that the wettability of hydrophobic slide could be rapidly altered by rhamnolipid biosurfactant. Once the rhamnolipid biosurfactant adsorbed on the hydrophobic glass surface lasting for 12 h，the contact angle could be dramatically decreased from 111.6° to 32.7°. The optimum dosage of rhamnolipid biosurfactant to extract oil from oilsands was 30%，resulting in the better oil displacement efficiency（i.e.， 84.83%）and the lower adhesion force（0.2 mN/m）. Furthermore，the free energy between deionized water and hydrophobic glass slide treated by rhamnolipid solution using the liquid-solid interface free energy formula was-140.2 mJ/m²，which was much lower than that between deionized water and original hydrophobic glass slide（-52.1 mJ/m²）. On the other hand，based on the molecular simulation，the rhamnolipid molecules could quickly adsorb on the hydrophilic surface via hydrogen bonds（strong interaction force），while both the electrostatic force and van der Waals force（weak interaction force）were the main interaction forces between rhamnolipid molecules and hydrophobic surface. Therefore，the adsorption binding energy between rhamnolipid molecules and hydrophilic surface（29.7 eV）was much higher than that between molecule and hydrophobic surface（12.2 eV），leading to the stronger interfacial competitive adsorption capacity of rhamnolipid molecules compared to asphaltenes. In other words，according to the free energy calculation and molecular simulation results，rhamnolipid molecules would easily anchor to the hydrophilic surface and“sweep off”the oil molecule，resulting in higher oil displacement efficiency. This paper provided an insight to construct an effective wetting control flooding system based on rhamnolipid biosurfactants.