In order to achieve green and efficient development of oil fields，a green，environmentally friendly and efficient biological nano-oil displacement system was prepared through combining the metabolites of Bacillus sp. 3096-3 with the reduction products of Shewanella sp. CD-8. The metabolites，reduction products and dispersion stability of the compound system were characterized by liquid chromatography-mass spectrometry（HPLC-MS/MS），infrared absorption spectroscopy（FT-IR），X-ray powder diffraction（XRD），scanning electron microscopy（SEM），particle size and Zeta potential analysis，and the emulsification performance and enhanced oil recovery ability were explored. The results showed that the bio-nano oil displacement agent system composed of lipopeptide biosurfactants and nano-iron particles had good dispersion stability，and the emulsification performance was positively correlated with the concentration of nano-iron particles. In the range of experimental conditions，the recovery rate increased with the increase of concentration of nano-iron particles and temperature，and the maximum recovery rate could reach up to 56.80% at the temperature 70 ℃ when the concentration of nano-iron particles was 100 mg/L. The recovery effect of bio-nano oil displacement agent system was better than that of single biosurfactant and nano-iron fluid. The prepared bio-nano oil displacement agent system provides a feasible method for the green development of oil fields.