A hemicyanine fluorescent dye was generated by a diamine-catalyzed, DNA-templated, aldol-type condensation from nonfluorescent precursors. Studies of reaction rate and yield as a function of catalyst structure indicated the diamine catalyst operates in a concerted mechanism on both reaction components. Our findings expand the scope of reactions that can be performed by DPC and demonstrate that de novo chemical synthesis of labels can be coupled to biological recognition events in a homogeneous format with essentially no background.