We report in this work real time characterization of enzyme-coenzyme binding by using surface plasmon resonance (SPR) and dual polarization interferometry (DPI) analyses. Results showed that diaphorase (DP) and lactate dehydrogenases (LDH) had distinct binding selectivity and preference over reduced and oxidized states of coenzyme NAD(H). On the basis of that, DP and LDH were chosen as indicator enzymes to distinguish the specific state of surface-bound NAD(H). The transformation between NADH and NAD+ during enzyme-catalyzed redox reactions was therefore transduced into variation in interaction signals as indicated via the binding status of the indicator enzymes as detected with both SPR and DPI. This real time molecule-specific detection strategy revealed quick and direct reflection of the state and reactivity of the coenzyme, promising a unique way of precise molecular interaction analysis.