A new avidin biotin assay was investigated on the basis of electrochemical detection. Dopamine labeled biotin (LB) was used as a bifunctional compound to mediate the oxidation of beta-nicotinamide adenine dinucleotide (NADH). The labeled biotin exhibited an effective electrocatalytic activity toward the oxidation of NADH as well as a specific binding with avidin. The labeled biotin's function as a mediator decreased when it bound with avidin, hence decreasing the electrode response. The detection of avidin and biotin was performed from the suppression of mediated current by the interaction between protein and ligands.