PURPOSE: To industrially effectively prepare amikacin which is an enzyme- resistant antibiotic, by suspending a specific kanamycin A derivative in a solvent, adding a formic acid aqueous solution in the presence of a catalyst such as Pt/C or the like, and eluting an adsorbed matter through an ion exchange resin column.

CONSTITUTION: 1-N-(L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyryl) 3,6'-di-N- benzyloxycarbonyl-kanamycin A is suspended in alcohol, a ketone, dimethylformamide, acetonitrile, an aliphatic ester, a hydrocarbon a chlorine- containing solvent or water or the like as a solvent in a ratio of 1:5-1:25, the almost equal quantities of a mixture of Pt/C, Pd/C and Raney Ni, and a catalyst of Pd/C or the like are added, and then 10-100% formic acid aqueous solution is slowly added at 0-100°C by 1:1, to obtain the reacted mixture including amikacin and kanamycin A, then the mixture is permeated through a ion exchange resin column, and is eluted by 2N NH3 solution, to separate a fraction including amikacin, and the solvent is evaporated under a reduced pressure, to obtain the amikacin.