Abstract: In this study, Fe₃O₄@ZrO₂ magnetic nanoparticles which were synthesized successfully were used as carrier for the highly selective enrichment of phosphopeptides from tryptic digest product of casein . Using N/P (molar ratio) of CPP and magnetic nanoparticles adsorbance as evaluating indicators, the influence of five variables which were the hydrolysis degree of casein, adsorption pH, adsorption time, adsorption temperature, and the initial concentration of phosphopeptides, on the ability of Fe₃O₄@ZrO₂ magnetic nanoparticles to isolate and enrich phosphopeptides was analyzed. The technical parameters of CPP enrichment were optimized. Results showed that, the optimized conditions of CPP enrichment were as below: Casein hydrolysis degree 22%, adsorption pH4.5, adsorption temperature 30 ℃, adsorption time 50 min, the initial concentration of phosphopeptides 50 mg/mL. Finally, under these conditions, the N/P (molar ratio) of CPP was 4.87, and the adsorbance of Fe₃O₄@ZrO₂ magnetic nanoparticles was 94.37. The bound CPP were eluted using NaOH (pH13) and the elution rate was 95% at least. Experiment results demonstrate that ZrO₂ coated-magnetic microspheres showed the excellent potential for selective enrichment of CPP, which would play an important role in the production of high quality and purity CPP.