Here, we report a novel route to prepare bioreactive surfaces on gold by the self-assembly of generation-three hydroxyl-terminated dendron thiols (G3-OH) and subsequent bridging reactions using generation two amine-terminated dendrimers (G2-NH2). It has been shown that G3-OH dendron thiols form a stable and uniform self-assembled monolayer on gold, which can be activated by the homobifunctional crosslinker N,N-disuccinimidyl carbonate (DSC). Subsequent derivatization of the activated monolayer via dendrimer bridging reactions with G2-NH2 enhances the stability, reactivity, and versatility of the prepared surface. Each step of the surface formation reaction has been monitored, and the resulting surface has been characterized by wetting, electrochemistry, scanning tunneling microscopy (STM), and infrared (IR) spectroscopy measurements. The reactivity of this surface was demonstrated by a Schiff base coupling reaction with 4-cyanobenzaldehyde, by immobilizing biotin molecules onto the peripheral amine groups using one of the conjugation methods, and by further binding avidin onto the biotinylated surface. We believe that the prepared bioreactive surface with a high density of amine groups will be useful for the immobilization of biological macromolecules for various biosensor applications, such as the fabrication of DNA microarrays and protein chips.