The pH is a crucial external factor affecting the structure and emulsification characteristics of proteins. The current study aimed to reveal the correlation between the secondary structure changes and tilapia protein isolate (TPI) emulsion stability under different pH (3.0–10.0) prepared by high-pressure homogenization. The results showed that TPI with significantly increased solubility and emulsifying properties when the pH keep away from the isoelectric point (pH 5.0). Meanwhile, TPI emulsions presented significantly enhanced stability (with decreased particle size, increased zeta potential, creaming index close to 0, and uniform dispersion of droplets) at pH 3.0 and 10.0. Interface-adsorbed protein mainly consists of a myosin-heavy chain and actin, and the secondary structure was significantly influenced by pH and high-pressure homogenization. The α-helix will be transformed into β-sheet and β-turn when pH is closer to pH 5.0. However, the high-pressure homogenization induced α-helix conversion to β-sheet. The correlation analysis revealed that emulsion stability is positively correlated with α-helix and negatively correlated with β-sheet. This work provides a deep insight into the correlation between secondary structure changes and the stability of TPI emulsion as affected by pH to offer an alternative way to enhance TPI emulsion stability.