Abstract: Lutein is a highly effective antioxidant, but its application is limited because its stability is easily affected by the environment. Tilapia protein isolate (TPI) is a high-quality animal protein with balanced amino acid ratio and high digestibility. However, due to the limitation of low solubility in water, its emulsifying activity and emulsifying stability are poor, and the application of TPI as emulsifier is limited. The pH value is the most obvious external factor that affects the functional properties of protein, and heat treatment is an essential operation in food processing. In order to improve the stability and bioavailability of lutein, tilapia protein isolate (TPI) was used as emulsifier, and TPI emulsions containing 200 μg/mL lutein were prepared by high pressure homogenization to explore the effects of heat treatment (70 °C, 30 min) on emulsion particle size, creaming index, lutein content, in vitro antioxidant activity and in vitro digestion at three pH values (3.0, 7.0 and 10.0). The results showed that after heat treatment, the particle size of TPI emulsion with lutein decreased, the emulsion droplets were evenly distributed, and the storage stability was enhanced. At pH 3.0, the stability of TPI emulsion loaded lutein was poor with lutein degradation, and the color of emulsion changed obviously. However, at pH 7.0 and 10.0, heat treatment of 70 °C significantly improved the stability of the emulsion without causing the degradation of lutein, and there was no delamination when stored at 4 °C for 28 days. After simulated gastrointestinal digestion in vitro, the release rate of free fatty acids was accelerated. After heat treatment, the bioavailability of lutein increased from 18.65%±1.06% to 35.69%±2.06% at pH 7.0. At pH 7.0, the free fatty acid release was 94.22%±0.60%, the bioavailability was 35.69%±2.06%, and the ATBS free radical scavenging ability was 59.17%±0.66%. The results showed that combined heat treatment under neutral and alkaline conditions can enhance the stability and bioavailability of lutein loaded TPI emulsion and protect the antioxidant activity of lutein, which provides a basis for the development and application of lutein-loaded TPI emulsion.