In cardiac muscle, binding of troponin (Tn) and tropomyosin (Tpm) to filamentous (F)‐actin forms thin filaments capable of Ca2+‐dependent regulation of contraction. Tpm binds to F‐actin in a head‐to‐tail fashion, while Tn stabilizes these linkages. Valuable structural and functional information has come from biochemical, X‐ray, and electron microscopy data. However, the use of fluorescence microscopy to study thin filament assembly remains relatively underdeveloped. Here, triple fluorescent labeling of Tn, Tpm, and F‐actin allowed us to track thin filament assembly by fluorescence microscopy. It is shown here that Tn and Tpm molecules self‐organize on actin filaments and give rise to decorated and undecorated regions. Binding curves based on colocalization of Tn and Tpm on F‐actin exhibit cooperative binding with a dissociation constant Kd of ~ 0.5 µm that is independent of the Ca2+ concentration. Binding isotherms based on the intensity profile of fluorescently labeled Tn and Tpm on F‐actin show that binding of Tn is less cooperative relative to Tpm. Computational modeling of Tn‐Tpm binding to F‐actin suggests two equilibrium steps involving the binding of an initial Tn‐Tpm unit (nucleation) and subsequent recruitment of adjacent Tn‐Tpm units (elongation) that stabilize the assembly. The results presented here highlight the utility of employing fluorescence microscopy to study supramolecular protein assemblies.