Alfalfa contains many important substances including flavonoids which play many diverse roles in plant. Alfalfa callus was induced on media containing 6- benzyl aminopurin (BA), α -Naphthalene acetic acid (NAA), 2, 4 Dichloro phenoxy acetic acid (2, 4-D) and kinetin. The best callus formation was obtained from the medium of 2 mgl^-1 2, 4- D + 1.5 mgl^-1 Kinetin; leaf was the best organ of alfalfa in production of fresh weight. The 2^nd subculture was higher in growth characters and flavonoids production as compared to the 1^st subculture. Flavonoid concentration was higher in shoot tip of 1^st subculture and leaves in the 2^nd subculture.