A new Valen Shiff base (C22H24N4O5) was synthesized using equivalent moles of o-vanillin and trimethoprim. At 298.15 K, the standard molar enthalpy of formation of the new compound was estimated to be ΔfHmΘ [C22H24N4O5(s), 298.15 K] = −(696.92 ± 1.67) kJ mol−1 by microcalorimetry. In particular, the interaction between the Shiff base and bovine serum albumin (BSA) has been investigated. It was proved that the fluorescence quenching of BSA by Shiff base is a result of the formation of a Shiff base-BSA complex. Quenching constants were determined using the Sterns–Volmer equation to provide a measurement of the binding site between Shiff base and BSA. The thermodynamic parameters ΔG, ΔH, and ΔS of the system at different temperatures were calculated. What is more, the distance r between donor (Trp. 213) and acceptor (Shiff base) was obtained. Finally, synchronous fluorescence spectroscopy data has suggested the association between Shiff base and BSA changed the molecular conformation of BSA.