A sugar-fatty acid ester, named glucose monooleate (GluO) was synthesized using commercial immobilized lipase B from Candida antarctica. A preparation of oil-in-water (O/W) emulsion using a combination of non-ionic surfactant mixture, i.e., GluO and Cremophor EL (CrEL), and different vegetable oils, i.e., coconut, sunflower, olive, palm and jojoba oils through phase inversion emulsification (PIE) was investigated. A baseline formulation was carried out to obtain an optimal ratio of surfactant-to-oil (S:O) and Cremophor EL-to-Glucose monooleate (CrEL:GluO) with coconut oil as the carrier oil. GluO proves to be an effective co-surfactant when a high amount of oil was incorporated. At 60:40 of S:O and CrEL:GluO, coconut and sunflower oil were emulsified into nano-sized oil droplets with the sizes (in diameter) of 36 ± 1.5 nm (PDI = 0.17 ± 0.05) and 42 ± 1.3 nm (PDI = 0.18 ± 0.07), respectively. In contrast, olive, palm and jojoba oils produced coarse emulsions with larger oil particles with the sizes of 238 ± 22, 295 ± 41 and 312 ± 63 nm, respectively. Furthermore, at 60:40 of S:O and CrEL:GluO, up to 0.98 mg of β-carotene (≈0.03% of total emulsion weight) can be encapsulated within coconut oil droplets of the O/W system with an average size below 100 nm. The emulsification process was investigated through Fourier-transform infrared (FTIR) spectroscopy, small-wide angle X-ray (SWAX) and optical polarizing microscopy (OPM) analyses. Possible effects of the surfactant mixture (CrEL-GluO) and different vegetable oils on the emulsion preparation were also discussed.