Abstract：The structures of two important intermediates in the synthesis of new kinds of aminoglycoside antibiotics were confirmed and the contents were determined by qNMR. The structures of the compound 1 and compound 2 were confirmed by 13C NMR, 1H NMR, 1H 1H-COSY, HMBC and MS. When maleic acid and sodium formate were respectively used as internal standards, and D2O was employed as solvent, the internal peak at δ6.23 for maleic acid and quantitative peaks at δ5.05 and δ5.66 for compound 1 and the internal peak at δ8.56 for sodium formate and quantitative peaks at δ2.00 and δ5.72 for compound 2 were obtained. The contents of compound 1 and compound 2 were calculated by comparing the response signal area of the sample and the internal standard. The compound 1 was 3-amino-3-deoxy-α-D-glucopyranosyl-(1→6)-[2,3,4,6-tetradeoxy-2,6-diamino]-α-D-erythro-hexopyranosyl-(1→4)]-1-N-[(2S)-4-amino-2-hydroxybutanoyl]-2-deoxy-D-streptamine and compound 2 was 3-amino-3-deoxy-α-D-glucopyranosyl-(1→6)-[2,3,4,6-tetradeoxy-2,6-diamino]-α-D-erythro-hex-3-enopyranosyl-(1→4)]-1-N-[(2S)-4-amino-2-hydroxybutanoyl]-2-deoxy-D-streptamine.The contents of compound 1 and compound 2 were 64.98% and 75.38%, respectively. The two compounds were substituted 2-deoxystreptamines and the key intermediates, which standards couldn't be purchased, in the synthesis of new kinds of aminoglycoside antibiotics could be determined by qNMR rapidly and accurately.