Nanoparticle-assisted enhanced oil recovery (Nano-EOR) has attracted intensive interest in the laboratory as a promising oil recovery technology. However, the nanoparticles’ stability and long-distance delivery of nanoparticles (NPs) in large-scale reservoirs are two main challenges. In this work, we developed a novel concept of in situ synthesizing NPs at the oil–water interface inside the reservoir for EOR instead of injecting presynthesized NPs from outside. The pore-scale flooding experiments show that EOR efficiencies for tertiary flooding were 6.3% without reaction (Case 3), 14.6% for slow reaction (Case 1), and 25.4% for relatively quick reaction (Case 4). Examination of the EOR mechanism shows that in situ produced SiO2 NPs in microchannels could alter the substrate wettability toward neutral wetting. Moreover, the produced NPs tended to assemble on the immiscible oil–water interface, forming a barrier toward interface deformation. As the reaction continued, excessive surface-modified NPs could also diffuse into aqueous brine and accumulate as a soft gel in the flowing path swept by brine. Collectively, these processes induced a “shut-off” effect and diverted displacing fluids to unswept areas, which consequently increased the sweep efficiency and improved the oil recovery efficiency. Auxiliary bulk-scale experiments also showed that the reaction-induced nanoparticle synthesis and assembly at an immiscible interface reduced the interfacial tension and generated an elastic oil–water interface.