The inhibitory effect of Picholine olive oil from Montpellier in Southern France on the chemical mediator release in type I allergy, using rat basophilic leukemia (RBL-2H3) cells, was investigated. Oil-in-water (O/W) emulsions prepared using Picholine olive oil showed an inhibitory effect on the chemical mediator release and decreased expressions of genes related to type I allergy in RBL-2H3 cells. We then measured the phenolic compounds present in Picholine olive oil using high-performance liquid chromatography and investigated some physical properties, such as droplet size, size distribution, viscosity, and surface tension of the resulting olive O/W emulsions. Our findings indicate that Picholine olive oil has high flavonoids content, especially apigenin, and the prepared emulsion of Picholine olive oil resulted in a considerable small size distribution, with an average droplet size of 170 nm.