This work investigated the stabilization mechanisms of annatto seed oil emulsions, rich in δ-tocotrienol, by employing biopolymers such as gum arabic (GA), whey protein isolate (WPI), modified starch (Hi-Cap and SF), polyethylene glycol (PEG) and inulin as emulsifiers/stabilizers. The effects of emulsification by high-intensity ultrasound were evaluated and compared to emulsification by mechanical stirring. The research in this study was focused on the ζ-potential and pH of the biopolymer suspensions, the dynamic tension on the interface between the annatto seed oil and water, the viscosity and rheological behavior, the size distribution and average diameter of the droplets, the optical microscopy and the creaming index of the emulsions. The steric stabilization mechanism predominated over electrostatic in the emulsions obtained with GA, WPI, Hi-Cap and SF. The stabilization of the emulsions with PEG or inulin was associated with a reduction in the oil droplet size and an increase in the viscosity of the continuous phase. In general, the emulsification efficiency achieved using ultrasound was greater than that of the mechanical stirring process for all biopolymers because it resulted in the formation of more stable emulsions over time as well as monomodal and narrow size distributions with smaller droplets, thus indicating uniform processing.