Delivery of drugs and active agents to human skin by formulation containing nanosystems have shown remarkable advance in recent nanotechnology research. The aim of current investigation is to study protein drug, extracted from medicinal leech tissue and evaluate an isotropic and kinetically stable nanoemulsion formulation, with least surfactant and co-surfactant concentrations also with optimal solubility and stability, for topical delivery. It is a fact that physical properties of oil phase has an impact on nanoemulsion formation and stabilization. In this research, various factors, such as oil types (olive oil and sesame oil) and oil content for their effects on particle size and stability of protein nanoemulsion were first investigated. Secondly, optimized formulation of protein nanoemulsion was characterized by droplet size and zeta potential analysis, Transmission electron microscopy (TEM), viscosity, pH, and refractive index. Thirdly, stability studies were done to select the best formulation. The results of our experiments showed that an increase in the concentration of olive oil and sesame oil led to a nanoemulsion with smaller size of droplets and with higher stability, respectively. However; slight variations in droplet size were observed in case of nanoemulsion with olive oil. As a result of various experiments, a Nanoemulsion with 25% olive oil was selected as optimized formulation owing to its much smaller droplet size (143.1 nm), lower polydispersity index, higher zeta potential (-33.3 mV). No considerable changes in droplet size, viscosity and pH occurred during a 30-day storage period at 4 °C. This procedure also suggested that our selected nanoemulsion was physically stable. Additionally, TEM revealed that particles were morphologically spherical. In conclusion, our analyses of the experiments proved the fact that nanoemulsions are promising novel formulations for protein drugs and can greatly enhance stability of protein drugs.