The interactive behavior of flavin adenine dinucleotide (FAD) with a bare glassy carbon electrode (GCE) and a Ru-modified GCE was investigated. The reduction of FAD at a GCE/ruthenium-modified GCE surface is a quasi-reversible, surface-controlled process, and our data implied that the attachment of FAD onto the surface is caused by nonspecific adsorption instead of covalent linkage, in which the adenine ring of FAD adopts a flat orientation on the GCE surface in neutral and dilute solutions in order to maximize the pi-pi stacking with the carbon surface and reorients to a perpendicular orientation as the surface gets more crowded. FAD desorption during the exchange with nicotinamide adenine dinucleotide (NAD(+)) is one order of magnitude slower than desorption in the absence of NAD(+), which indicates a strong interaction between FAD and NAD(+). General knowledge of the interactive behavior of NAD(+) on a FAD-adsorbed GCE provides useful information for the design of a modified electrode surface for the generation of NADH from NAD(+).