Abstract: In this paper, a novel casein phosphopeptide fractionation process was proposed based on the calcium chelating properties of casein phosphopeptide and different casein phosphopeptide fractions with different chelating abilities and enhanced purity were produced using different calcium ion concentrations Three casein phosphopeptide-calcium chelate fractions (CPP1-Ca, CPP2-Ca and CPP3-Ca) were precipitated hierarchically from the trypsin digest of casein by increasing the Ca²⁺ addition step by step in an ethanol solution with a concentration of 55% (v/v), and PBS was used to decalcify to obtain the corresponding casein phosphopeptides (CPP1, CPP2 and CPP3). The physicochemical properties and calcium uptake characteristics of the three casein phosphopeptide-calcium chelate fractions were analyzed and compared with those of the peptide-calcium chelate fraction (CPP-Ca) obtained by the conventional calcium-ethanol precipitation method as a control. The results showed that among the three casein phosphopeptide-calcium chelate fractions, CPP3-Ca contained the highest purity and calcium chelating amount of 89.42% and 69.59 mg/g, respectively, which were significantly higher than those of the ungraded CPP-Ca of 83.77% and 55.05 mg/g. The analysis of the amino acid composition and the N/P (molar ratio) revealed that the CPP3-Ca contained a higher phosphatidylserine group, which in turn bound a higher amount of Ca²⁺. which in turn binds more Ca²⁺. Analysis of the calcium holding capacity of different casein phosphopeptide fractions showed that CPP3 was able to better delay and prevent the formation of calcium phosphate precipitation, and had a higher calcium holding capacity. Meanwhile, CPP3-Ca has higher calcium solubility in the simulated intestinal environment, indicating that the in vitro bioavailability of the graded casein phosphopeptide-calcium chelates is improved.