Transforming growth factor-β (TGF-β) binds with two transmembrane serine/threonine kinase receptors, type II (TβRII) and type I receptors (TβRI), and one accessory receptor, type III receptor (TβRIII), to transduce signals across cell membranes. Previous biochemical studies suggested that TβRI and TβRIII are preexisted homo-dimers. Using single-molecule microscopy to image green fluorescent protein-labeled membrane proteins, for the first time we have demonstrated that TβRI and TβRIII could exist as monomers at a low expression level. Upon TGF-β1 stimulation, TβRI follows the general ligand-induced receptor dimerization model for activation, but this process is TβRII-dependent. The monomeric status of the non-kinase receptor TβRIII is unchanged in the presence of TGF-β1. With the increase of receptor expression, both TβRI and TβRIII can be assembled into dimers on cell surfaces.