•A novel calcium-binding peptide was identified from tilapia scale hydrolysates.•The amino acid sequence of the peptide was DGDDGEAGKIG.•The stability of the peptide–calcium complex was evaluated in vitro.•The calcium bioavailability of the peptide was evaluated in vivo.A calcium-binding peptide was successfully purified from tilapia (Oreochromis niloticus) scale protein hydrolysate by hydroxylapatite affinity (HA), gel filtration and reverse phase high-performance liquid chromatography (RP-HPLC). This peptide was sequenced as DGDDGEAGKIG (N- to C-terminal, MW = 1033 Da) based on MALDI-TOF-MS/MS. There was no significant difference in body weight gain, serum P content or alkaline phosphatase (ALP) level in Ca-deficient rats after supplementation with TSPH–Ca (tilapia scale protein hydrolysate calcium complex), CPP–Ca (casein phosphopeptide calcium complex), CaCO3 or saline (control). However, the apparent absorptivity of calcium, serum and femur Ca content, femur bone mineral density and strength in the rats fed with TSPH–Ca were significantly higher than those of rats in the control and CaCO3 groups (P < 0.05) and were similar to those from the CPP–Ca group (P > 0.05), indicating that TSPH possesses the potential to serve as an alternative for CPP.