To differentiate commercial bifidobacteia for Bifidobacterium lactis Bb-12, Bif. longum Bb-536 and Bif. infantis Maeil-K9, we studied the various carbon source, the nitrogen sources and antibiotics. Amygdalin and fructose were good candidates for carbon sources, and tryptone was suitable for nitrogen sources to design a new selective media for three commercial bifidobacteria. In the case of the amygdalin-containing medium as carbon sources, Bif. lactis Bb-12 and Bif. infantis Maeil-K9 showed good growth, and in fructose-containing medium, Bif. longum Bb-536 showed good growth. In antibiotics resistance study, the addition of 1 mg/L doxycyclin was very effective for differentiation of each bifidobacteria. Doxycyclin did not affect the growth of Bif. lactis Bb-12 and Bif. infantis Maeil-K9, but Bif. longum Bb-536 was completely inhibited by doxycyclin. Finally to confirm the selection capability of newly designed selective media, temperature-shocked bifidobacteria were cultured on them. As the results, fructose or doxycyclin containing medium showed for high growth for temperature-shocked bifidobacteria, but amygdalin containing medium showed low growth of temperature-shocked bifidobacteria.