Abstract: The objective of the present work was to fabricate ovotransferrin–gum arabic particles with desirable properties as Pickering stabilizers. Complexation of ovotransferrin (OVT) with gum arabic (GA) was investigated using turbidimetric titrations, and OVT–GA complexation proved to be an effective method to assemble hardcore food-grade nanoparticles. Preparation of OVT–GA particles was optimized by controlling biopolymer ratios and pH values, and uniform OVT–GA nanoparticles with mean particle size of 175.8 ± 1.3 nm and air-water contact angle of 82.6° were prepared at r = 3:1 and pH 3.2. Involvement of electrostatic interaction, hydrophobic interaction and hydrogen bonding in nanoparticle formation was verified through titrations in the presence of destabilizing agents. Interfacial tension measurements revealed that OVT–GA nanoparticles could effectively reduce interfacial tension. Afterwards, OVT–GA nanoparticles were employed to prepare surfactant-free Pickering emulsions. Visual observation indicated that Pickering emulsions stabilized by OVT–GA nanoparticles possessed superior emulsified phase volume fraction and storage stability. Confocal laser scanning microscopy results indicated that OVT–GA nanoparticles were located at the oil-water interface. Microstructures of emulsions were dependent on the concentrations of applied particles and oil fraction. Rheological measurements of Pickering emulsions demonstrated that increasing either particle concentration or oil fraction could lead to an increase of storage modulus and viscosity.