The　bioavailability　of　dietary　ionised　calcium　is　affected　by　intestinal　basic　environment.　Calcium-binding　peptides　can　form　complexes　with　calcium　to　improve　its　absorption　and　bioavailability.　The　aim　of　this　study　was　focused　on　isolation　and　characterisation　of　a　calcium-binding　peptide　from　whey　protein　hydrolysates.　Whey　protein　was　hydrolysed　using　Flavourzyme　and　Protamex　with　substrate　to　enzyme　ratio　of　25　:　1　(w/w)　at　49　°C　for　7　h.　The　calcium-binding　peptide　was　isolated　by　DEAE　anion-exchange　chromatography,　Sephadex　G-25　gel　filtration　and　reversed　phase　high-performance　liquid　chromatography　(RP-HPLC).　A　purified　peptide　of　molecular　mass　204　Da　with　strong　calcium　binding　ability　was　identified　on　chromatography/electrospray　ionisation　(LC/ESI)　tandem　mass　spectrum　to　be　Glu-Gly　(EG)　after　analysis　and　alignment　in　database.　The　calcium　binding　capacity　of　EG　reached　67·81　μg/mg,　and　the　amount　increased　by　95%　compared　with　whey　protein　hydrolysate　complex.　The　UV　and　infrared　spectrometer　analysis　demonstrated　that　the　principal　sites　of　calcium-binding　corresponded　to　the　carboxyl　groups　and　carbonyl　groups　of　glutamic　acid.　In　addition,　the　amino　group　and　peptide　amino　are　also　the　related　groups　in　the　interaction　between　EG　and　calcium　ion.　Meanwhile,　the　sequestered　calcium　percentage　experiment　has　proved　that　EG-Ca　is　significantly　more　stable　than　CaCl2　in　human　gastrointestinal　tract　in　vitro.　The　findings　suggest　that　the　purified　dipeptide　has　the　potential　to　be　used　as　ion-binding　ingredient　in　dietary　supplements.　©　2015　Proprietors　of　Journal　of　Dairy　Research　.