Expanding on theoretical work by Heinze et al.1, an optical setup to experimentally realize three-colour fluorescence cross-correlation spectroscopy (3C-FCCS) was designed. This instrument simultaneously collects and correlates fluorescence data from three colour channels, allowing real-time tracking of three-coloured species in complex solutions. To examine 3C-FCCS theory, a nanobarcode test particle, which physically linked three fluorophores was selected and characterized. These nanobarcode particles, capable of producing a triple cross-correlation decay, facilitated proof-of-concept experiments. 3C-FCCS was shown to be capable of measuring the concentration of nanobarcode particles in solution exclusively containing nanobarcode particles and solution containing nanobarcodes outnumbered by background quantum dots at a ratio of 800:1. 3C-FCCS was used to investigate a simple DNA-based nanostructure. These experiments illustrate that 3C-FCCS is a compelling technique with a plethora of potential applications, ranging from investigating hierarchical nanoassembly to biological reactions.