In this study, a Schiff base derived from a heterocyclic moiety was synthesized and characterized. The in vitro binding behaviour of this ligand with β‐casein (β‐CN) was investigated using biophysical techniques. For evaluation, thermodynamics variables of interactions between the Schiff base ligand and β‐CN, such as fluorescence at different temperatures, were measured. The results showed that the Schiff base ligand possessed considerable associated binding to β‐CN and that the procedure was enthalpy driven. The β‐CN conformation was also changed to give a further unfolded structure. Fluorescence resonance energy transfer was used to estimate the interval between donor (β‐CN) and acceptor (Schiff base ligand). All these experimental results proposed that β‐CN might act as carrier protein for the Schiff base ligand to deliver it to the target molecules.