Abstract: In order to improve the utilization of pearl, calcium supplement with high bioavailability was produced. Pearl peptide was prepared from pearl powder and chelated with calcium. The preparation process was optimized based on the chelation yield and calcium content in pearl peptide chelated calcium. The changes of chelation were compared by ultraviolet-visible absorption spectroscopy (UV-vis), fourier infrared spectroscopy (FTIR), circular dichroism (CD), X-ray diffraction (XRD), difference scanning calorimetry (DSC) and scanning electron microscopy (SEM) analysis. The results showed that pearl protein was obtained from pearl powder by acid hydrolysis and repeat centrifugation. The pearl protein was hydrolyzed by Bacillus licheniformis proteinase (BLP) to obtain pearl peptide. The final yield of pearl peptide chelated calcium was 27.24%±1.29%, and the calcium content in pearl peptide chelated calcium was 48.75%±1.20%. The structure of the pearl peptide and pearl peptide chelated calcium were then analyzed. It was found that both amino and carboxyl groups of pearl peptide were involved in the chelation reaction. And the secondary structure of pearl peptide chelated calcium changed from disordered structure to ordered and compact structure. The crystallinity increased greatly and a new substance with more stable structure was formed after the chelation reaction. And a significant aggregation was occurred in calcium-chelating peptide.