This paper focuses on the preparation and bioactivity of Ca²⁺ chelate of hydrolysate which from the waste fish proteins through compound enzymolysis. The optimum formation conditions of the hydrolysate chelating Ca²⁺ are 5% of DH, 7.0 of pH, 293 K of temperature and 15 min of chelating time for FM (material not being defatted). Three types of hydrolysate chelating Ca2+ including CA (deposit after chelating), CB (deposit in 50% of absolute alcohol solution) and CC (deposit in 80% of absolute alcohol solution) were obtained through fractional precipitation with absolute alcohol from FM. Structure analysis through IR revealed that Ca2+ had been combined strongly with amino-group and carboxyl-group in each of chelates and each of Ca²⁺ could form 2 five-memberd ring structures in CA, however, Ca²⁺ had been only combined strongly with carboxyl-group in CB and CC. Among all of the chelates, the CA possesses the most effective antioxidative activity and the activity is 94% as high as that of a-tocopherol, and only CC possesses the most effective antibacterial activity against E. coli, S. aureus and B. subtilis and the activity depends on the size of the dissolvability in aqueous system and the strength of the electron buffering capability of the electron relaying system for CC. These researches provide a more effective way for the utilization of waste fish proteins and the essential information for the enzymatically hydrolyzed waste fish protein chelating Ca²⁺ as additive for food industry.