Homogeneous-type enzyme-linked competitive binding assays utilizing the synthetic enzyme-biotin and avidin-riboflavin conjugates are developed for the detection of riboflavin as well as its binder protein. The activity of the enzyme-biotin conjugate is inhibited in the presence of the avidin-riboflavin conjugate, and the observed inhibition is reversed in an amount dependent on the concentration of riboflavin binding protein (RBP) added. Upon additions of free riboflavin to the mixture, activity is reinhibited in an amount proportional to the riboflavin concentration. Three different enzymes are examined as the labels: glucose-6-phosphate dehydrogenase, adenosine deaminase, and alkaline phosphatase. The catalytic activity of these enzymes, when conjugated with biotin, is shown to be inhibited to a significant degree (> 90%) by the binding of the avidin-riboflavin conjugate, and reversed upon additions of RBP.