Abstract: In response to the overlooked influence of precursor molecular structure on interfacial performance in the application of amphiphilic carbon dots (CDs) for enhanced oil recovery (EOR), this study synthesized nitrogen-doped CDs (NCDs, FG and TA series) using biomass-derived precursors via carbonization, amidation, quaternization, and alkylation. The relationships between precursor structure, surface functionality, interfacial behavior, and oil displacement performance were systematically investigated. TA-derived NCDs exhibited higher surface polarity and amphiphilicity due to abundant carboxyl groups, while increasing alkyl chain length enhanced hydrophobicity and suppressed surface defects. TA-NCDs-L16 showed the best interfacial properties, with a critical micelle concentration (CMC) of 0.104 g/L, γCMC of 24.71 mN/m, and zeta potential of +67.80 mV. Under NaCl concentrations ranging from 0 to 12 wt%, the oil–water interfacial tension decreased to a minimum of 0.00151 mN/m, and the contact angle dropped to 16.3°, indicating excellent salt tolerance and wettability reversal capability. In low-permeability core flooding tests, TA-NCDs-L16 achieved a significantly enhanced final oil recovery of 60.42%, with a 27.26% increase in recovery and a 38.71% reduction in injection pressure. The improved EOR performance was attributed to ultra-low interfacial tension, the formation of high-density polar adsorption layers, and nanoscale size effects that enabled efficient pore-throat penetration and fluid redistribution, thereby facilitating the detachment and mobilization of residual oil. In high-salinity formation water containing Ca2+/Mg2+ and under elevated temperatures (50–90 °C), further evaluation confirmed that the amphiphilic NCDs maintained strong interfacial activity and sustained wettability reversal. TA-NCDs-L16 retained an ultra-low interfacial tension (∼0.002 mN/m) and stable wettability regulation even after 240 h of thermal aging at 80 °C, while core flooding still exhibited significant reductions in injection pressure and enhancements in oil recovery. This study clarifies the correlation among precursor structure, functional group configuration, interfacial behavior, and oil displacement efficiency, providing theoretical guidance and material design concepts for the development of carbon-based amphiphilic nanofluids in low-permeability reservoir applications.