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 Delta H-f(m)Theta [C22H24N4O5(s), 298.15K] = -(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 Delta G, Delta H, and Delta S 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. (C) 2013 Elsevier B.V. All rights reserved.