The interactions of D-glucose, D-fructose, D-galactose, and sucrose with disodium-adenosine-5′-monophosphate (Na 2AMP), disodium-2-deoxyadenosine-5′-monophosphate (Na 2dAMP), disodium-adenosine-5′-triphosphate (Na 2H 2ATP), disodium-guanosine-5′-monophosphate (Na 2GMP), disodium-2-deoxyguanosine-5′-monophosphate (Na 2dGMP), and disodium-guanosine-5′-triphosphate (Na 2H 2GTP) are investigated in aqueous solution at physiological pH with sugar/nucleotide ratios (r) of 1 10 , 1 2 , 1, and 2. Fourier transform infrared (FTIR) difference spectroscopy is used to establish a correlation between spectral changes and sugar binding mode, nucleotide confirmation, and sugar anomeric structure, as well as structural properties of sugar-nucleotide complexes in aqueous solution. Spectroscopic evidence showed that at low sugar concentration r = 1 10 , carbohydrate interaction (H-bonding) is mainly through phosphate (α-, β-, and γ-PO 2 − of the triphosphate chain or −PO 3 2− of monophosphate) groups, while at higher sugar content (r > 1 10 ) , sugar interaction (H-bonding) extends to base donor sites (adenine NH 2, N-1, and N-7 groups and guanine O-6 and N-7 atoms). The sugar-phosphate binding is quantitatively larger for the phosphate groups (30–60% ± 5%) than for the bases donor atoms (10–35% ± 3%). Evidence for sugar-phosphate interaction (H-bonding) comes from major spectral changes (intensity alterations and shiftings) of the antisymmetric and symmetric stretching vibrations of the PO 3 2− and different PO 2 − groups, located at 1250-900 cm −1, whereas sugar base binding (H-bonding) is characterized by the spectral alterations (intensity variations and shiftings) of the pyrimidine and imidazol in-plane vibrations at 1700-1400 cm −1. Evidence for sugar complexation also comes from the spectral shiftings of the carbohydrate OH stretchings (3500-3200 cm −1) and OH bendings (1450-1200 cm −1), as well as the CO and CC stretching frequencies (1100-900 cm −1). the ribose and deoxyribose moieties of the free ATP, AMP, and dAMP with C2−endo/anti conformation (with marker infrared band at 820–825 cm −1) exhibit no alteration upon sugar complexation. Similarly, the ribose and deoxyribose of guanosine-nucleotides with C2′-endo/anti- and C3′-endo/anti sugar puckers with marker IR bands at 800–822 cm −1 show no major conformational alteration on carbohydrate complexation. The sugar interaction occurs via both α- and β-anomeric structures.