Abstract: Janus amphiphilic polymer nanosheets (JAPNs) with anisotropic morphology and distinctive performance have aroused widespread interest. However, due to the difficulty in synthesis and poor dispersion stability, JAPNs have been scarcely reported in the field of enhancing oil recovery (EOR). Herein, a kind of organic-based flexible JAPNs was prepared by paraffin emulsion methods. The lateral sizes of JAPNs were ranging from hundreds of nanometers to several micrometers and the thickness was about 3 nm. The organic-based nanosheets were equipped with remarkably flexible structures, which could improve their injection performance. The dispersion and interfacial properties of JAPNs were studied systematically. By modification of crosslinking agent containing multiple amino groups, the JAPNs had excellent hydrophilicity and salt resistance compared with conventional inorganic or composite nanosheets. The settling time of nanosuspension with NaCl and CaCl2 at a low salinity of 1000 mg/L was over 240 h. The value could also remain 124 h under the salinity of 10,000 mg/L NaCl. With the dual functionalities of Janus amphiphilic nature and nanoparticles' Pickering effect, JAPNs could change rock wettability and form emulsions as “colloidal surfactants”. In particular, a new technology called optical microrheology was pioneered to explore the destabilization state of nanosuspensions for the first time. Since precipitation lagged behind aggregation, especially for stable suspension systems, the onset of the unstable behavior was difficult to be detected by conventional methods, which should be the indicator of reduced effectiveness for nanofluid products. In addition, the oil displacement experiments demonstrated that the JAPNs could enhance oil recovery by 17.14% under an ultra-low concentration of 0.005% and were more suitable for low permeability cores. The findings can help for a better understanding of the material preparation of polymer nanosheets. We also hope that this study could shed more light on the nanoflooding technology for EOR.