π–π Interaction is a direct attractive non-covalent interaction between aromatic moieties, playing an important role in DNA stabilization, drug intercalation, etc. Aromatic rings interact through several different conformations including face-to-face, T-shaped, and offset stacked conformation. Previous quantum calculations indicated that T-shaped and offset stacked conformations are preferred for their smaller electron repulsions. However, substitution group on aromatic ring could have a great impact on π–π interaction by changing electron repulsion force between two rings. To investigate π–π interaction between ligand and aromatic side chain of protein, Brookhaven Protein Data Bank was analyzed. We extracted isolated dimer pairs with the aim of excluding multiple π–π stacking effects and found that T-shaped conformation is prevalent among aromatic interaction between phenyl ring of ligand and protein, which corresponds with the phenomenon of Phe–Phe interactions in small peptide. Specifically, for the non-substitution model, both Phe–Phe and Phenyl–Phe exhibit a favored T-shaped conformation whose dihedral angle is around 50°–70° and centroid distance is between 5.0 and 5.6 Å. However, it could be changed by substituent effect. The hydroxyl group could contact in the case of Tyr–Tyr pairs, while they point away from phenyl plane in Phe–Tyr pairs.