Phosphatidylserine (PS) is a major anionic phospholipid constituent of membrane bilayers, which is specifically enriched in the cytoplasmic leaflet, has functions of regulating the intracellular signaling pathways of neuronal survival and differentiation, and acts as a neurotransmitter to control the activity of neurons. Oil-in-water (O/W) emulsions could improve the bio-availability of PS. Thus, there is a high level of interest in PS emulsion because of its purported health benefits. However, because of high viscosity and poor fluidity, it remains difficult to make the emulsion. A detailed analysis with suited biophysical methods would help to better understand the processes on a molecular level. Therefore, the main aim of the present study was to engineer and characterize a stable O/W phosphatidylserine emulsion. Furthermore, the effect of emulsifiers mixture, whey protein isolate (WPI), and Tween 80 (T80), as well as the oil phase was systematically evaluated. The key parameters were the chain length and the degree of nonsaturation (sunflower oil, a long-chain triglycerides [LCTs] or a medium-chain triglycerides [MCTs]). Small droplets of emulsions could be obtained by adjusting the type of emulsifier and the LCT/MCT ratio. A stable PS emulsion characterized by a smaller droplet size, higher negative zeta-potential, lower centrifugal stability constant, and longer storage time was produced by MCTs T80 (2.0%, w/w) with T80 (2.0%, w/w) as the emulsifier, and by LCTs with the WPI (0.5%, w/w)—T80 (1.5%, w/w) as the emulsifier, respectively. The PS emulsion with LCTs exhibited higher viscosity, when compared to the emulsion made by MCT at the same emulsifier concentration, while all emulsions exhibited a shear thinning behavior. The microstructure images revealed that the PS emulsions produced by MCTs and T80 (2.0%, w/w) or WPIs (0.5%, w/w)—T80 (1.5%, w/w) mixed with LCTs can form specific uniform networks, in order to prevent flocculation. After 28 days of storage, no visual phase separation was observed in the emulsions, except for the PS emulsion with the WPI (2.0%, w/w). It was concluded that the characteristics of the interfacial layer of particles in the PS emulsion system were not only dependent on the proportion of the applied emulsifiers, but also dependent on the oily phase features. These findings may provide indications for choosing the suitable process parameters when a stable PS emulsion is produced.