In this study, with grass fish bones as the substrate, after flavourzyme treatment, and fermentation with Leuconostoc mesenteroides, a fermentation solution with a high content of soluble calcium was obtained. High performance liquid chromatography and GC-MS analysis indicated that free calcium (11.29 mmol L-1) in the fermentation solution was composed of calcium lactate (3.89 mmol L-1), calcium acetate (6.21 mmol L-1), calcium amino acids and small peptide calcium. Animal experiments show that the fermentation solution of grass fish bones could promote the growth and development of calcium-deficient rats. Complex organic calcium could be well absorbed and utilized by rats so that serum calcium, alkaline phosphatase levels, femur weight and other indicators in calcium-deficient rats could be returned to normal levels. The fermentation solution of grass fish bones can avoid the waste of aquatic proteins and fish bone calcium, and it exhibited high calcium bioavailability. Therefore, the fermentation solution of grass fish bones might be used as a new efficient calcium supplement.