The binding of [Ru(PAN)(PPh3)2(ISN)]Cl (PAN = 1-(2'-Pyridylazo)-2-naphtholate) to bovine serum albumin (BSA) was investigated by spectroscopic techniques. According to analysis of the results from the Stern-Volmer equation, the ruthenium complex is able to quench the fluorescence intensity of BSA via a dynamic mechanism. The thermodynamic parameters were calculated (ΔH = 30.3 kJ mol-1; ΔS = 195.4 J mol-1 K-1), indicating that hydrophobic force is the main interaction driving force. The site marker competitive experiments revealed that the binding site of ruthenium complex was in the sub-domain IIA of BSA. FTO glass with a film of BSA-[Ru(PAN)(PPh3)2(ISN)]Cl was used as an ascorbic acid sensor. The linear range of the modified electrode was between 1 and 8 × 10-6 mol L-1.