In this study, spherical aggregates were prepared by ovalbumin (OVA) and sodium tripolyphosphate at pH 7 and 65 °C for 0, 6, 12, 18, 24 h, and the properties of phosphorylated OVA (P-OVA) spherical aggregates forming and stabilizing Pickering O/W emulsions were studied. Results showed that the P-OVA heated for 12 h showed the highest surface hydrophobicity, which was demonstrated by static contact angles. The fluorescence intensity results showed that the more chromophore were exposed to the surrounding microenvironment because of protein conformational transitions. The dynamic tracking of P-OVA spherical aggregates was performed by transmission electron microscope, and the observation demonstrated that spherical aggregate formed after 12 h of heating were closely distributed and well dispersed, showing a very dense network shape. Besides, the emulsifying ability of P-OVA spherical aggregates was demonstrated by dynamic interfacial tension, which was due to the hydrophobic interaction in protein aggregates enabling the formation of stable Pickering nano-droplets. The Pickering emulsion stabilized by P-OVA spherical aggregates showed better salt ion stability (0–100 mmol/L NaCl) and thermal stability (0–95 °C) as compared to emulsions prepared by the unheated sample. These findings demonstrate the unique properties of spherical aggregates based on P-OVA for preparing Pickering emulsions, which can provide theoretical guidance in the food industry system.