The interaction of a water-soluble dinuclear nickel(II) complex, [Ni2(EGTB)(CH3CN)4](ClO4)4·4H2O (EGTB = ethylene glycol-bis(β-aminoethyl ether) N,N,N',N'-tetrakis(2-benzimidazoyl)) (1), and bovine serum albumin (BSA) was investigated under physiological conditions using fluorescence, synchronous fluorescence, UV-vis absorption and circular dichroism (CD). The experimental results suggested that the nickel(II) complex could bind to BSA with binding constant (K) ~ 10(4) M(-1) and quench the intrinsic fluorescence of BSA through a static quenching mechanism. The thermodynamic parameters, ΔG°, ΔH°, and ΔS°, calculated at different temperatures, indicated that the binding reaction was spontaneous and electrostatic interactions played a major role in this association. Based on the number of binding sites, it was considered that one molecule of complex 1 could bind to a single site or two sites of the BSA molecule or the two binding modes coexisted. In view of the results of site marker competition experiments, the reactive sites of BSA to complex 1 mainly located in subdomain IIA (site I) and subdomain IIIA (site II) of BSA. Moreover, the binding distance, r, between donor (BSA) and acceptor (complex 1) was 5.13 nm according to Förster nonradiation energy transfer theory. Finally, as shown by the UV-vis absorption, synchronous fluorescence and CD, complex 1 could induce conformation and microenvironmental changes of BSA. The results obtained herein will be of biological significance in toxicology investigation and anticancer metallodrug design.