Actin filaments are involved in most cellular processes including determination of cell shape, cell migration, cell division, membrane function and intracellular transport.  This enormous functional specialisation is associated with differences in filament organisation, dynamics and interaction with actin-binding proteins.  Tropomyosins are key players in this dynamic regulation of the functions of actin filaments but the molecular mechanisms underlying the assembly of tropomyosin strands on actin filaments are not known.  Here we developed a single-molecule approach to follow the dynamics of the process at the molecular level. Actin filaments are nucleated on surfaces and aligned by the flow inside a microfluidic channel for observation by total internal reflection micorscopy. We then visualise the competitive binding and the dissociation of different fluorescent tropomyosin isoforms in real time. Our observations suggest that stable actin-tropomyosin filaments require a mechanism for coordinated assembly.