Abstract: Soybean protein was a high-quality plant-based alternative protein, yet its calcium content was lower than animal protein. This study measured the calcium-binding ability of soybean protein under different extraction, heat treatment, and enzymatic hydrolysis conditions. We compared the differences in calcium-binding ability, subunit composition, secondary structure, and hydrophobicity of soybean protein under four different preparation methods. The relationship between the calcium-binding ability and the structure of soybean protein was discussed. The results showed that the binding of soybean protein and calcium ions was related to both the protein's secondary structure composition and its hydrophobicity. Heat treatment and enzymatic hydrolysis unfolded the secondary structure, leading to the enhancement of hydrophobicity and the exposure of the calcium-binding sites. Enzymolysis led to the aggregation of small molecular weight proteins, causing a spatial restructuring of the protein. These changes resulted in a 53% increase in bound calcium compared to heat treatment. The heat-enzyme combined treatment was the most effective among various treatments, which could increase the Ca²⁺ of the protein by about 2 folds and decrease the precipitation rate by 43%. Optimal soybean protein with high calcium-binding ability preparation involves dissolution at pH8 and 25 ℃, with acid-precipitated proteins dissolved in four times water at pH 8. Subsequent steps include heating at 100 ℃ for 30 minutes, and enzymatic treatment using 1000 U/g flavor protease for three hours.