This study aimed to develop a long-term stable nanoemulsion of a perfluorocarbon (PFC)-based artificial red blood cell substitute. We analyzed the roles of the surfactant Tween 80 and the stabilizer Casein sodium by preparing formulations with and without 2% Casein sodium and adding various concentrations of Tween 80 (1%, 2%, 3%, 5%, 7.5%, 10%) to each. Particle size changes were observed over 21 days using dynamic light scattering (DLS). Our findings revealed that formulations lacking Casein sodium showed a rapid and significant increase in particle size across all Tween 80 concentrations, indicating an unstable state. Notably, the particle growth rate reached 409.1% at a 10% Tween 80 concentration. In contrast, formulations containing 2% Casein sodium showed a remarkably lower growth rate, with the lowest increase of 67.5% at a 3% Tween 80 concentration. This suggests that the amphiphilic structure of the protein induces interfacial tension between the aqueous phase and the oil droplets, thereby stabilizing the emulsion system. Furthermore, initial particle size analysis showed that the 5% Tween 80 concentration consistently resulted in the smallest particle size, regardless of the presence of Casein sodium. This indicates that 5% Tween 80 is the optimal ratio for nanoemulsion preparation. In conclusion, including Casein sodium with 3–5% Tween 80 is the most effective way to develop a stable artificial red blood cell substitute.