Both the binding and releasing of ferric ions in C-, and N-terminal binding sites of human serum transferrin are different. To understand the difference here the interactions of aluminum with the ligands containing phenolic group(s), including 8-hydroxyquinoline, salicylic acid, N, N′-di(2-hydroxybenzyl)ethylenediamine- N, N′-diacetic acid, N, N′-ethylenebis[2-( o-hydroxyphenolic)glycine], and human serum apotransferrin, respectively, are investigated by using UV difference and fluorescence spectra methods in 0.1 M N-2-hydroxyethylpiperazine- N-2-ethanesulfonic acid at pH 7.4. Aluminum binding produces a UV difference peak near 235 nm that is characteristic of phenolic groups binding to aluminum. The peak at 235 nm has been used to determine conditional binding constants of log K Al–HBED=8.88±0.74 and log K Al–EHPG=9.38±0.03. However, the effects of aluminum binding on the fluorescence intensity of N, N′-ethylenebis[2-( o-hydroxyphenolic)glycine], salicylic acid and N, N′-di(2-hydroxybenzyl) ethylenediamine- N, N′-diacetic acid, 8-hydroxyquinoline are disparate, the former showing a decrease and the latter an increase. At pH 7.4, there is N⋯H–O type intramolecular hydrogen bond in 8-hydroxyquinoline, N, N′-di(2-hydroxybenzyl) ethylenediamine- N, N′-diacetic acid and O⋯H–O type intramolecular hydrogen bond in salicylic acid, N, N′-ethylenebis[2-( o-hydroxyphenolic)glycine]. The effects of salts on the fluorescence intensity of the ligands containing phenolic group(s) show that fluorescence emission increases with the breaking of an N⋯H–O type intramolecular hydrogen bond and fluorescence emission decreases with the breaking of an O⋯H–O type intramolecular hydrogen bond. Fluorescence titrations of apotransferrin and both forms of monoferric transferrin with aluminum indicated that there is O⋯H–O type intramolecular hydrogen bonds for the phenolic groups of Tyr426 and Tyr517 in the C-terminal binding site. While N⋯H–O type intramolecular hydrogen bonds are found for the phenolic groups of Tyr95 and Tyr188 in the N-terminal binding site.