In-solution conjugation is the most commonly used strategy to label peptides and proteins with fluorophores. However, lack of site-specific control and high costs of fluorophores are recognised limitations of this approach. Here, we established facile access to grams of Cy5-COOH via a two-step synthetic route, demonstrated that Cy5 is stable to HF treatment and therefore compatible with tert-butyloxycarbonyl solid phase peptide synthesis (Boc-SPPS), and coupled Cy5 to the N-terminus of α-conotoxin RgIA while still attached to the resin. Folding of the two-disulfide containing Cy5-RgIA benefitted from the hydrophobic nature of Cy5, resulting in only the globular disulfide bond isomer. In contrast, wild-type α-RgIA folded into the inactive ribbon and bioactive globular isomer under the same conditions. Labelled α-RgIA retained its ability to inhibit acetylcholine (100 µM)-evoked current reversibly with an IC50 of 5.0 nM (Hill coefficient = 1.7) for Cy5-RgIA and an IC50 of 1.6 (Hill coefficient = 1.2) for α-RgIA at the α9α10 nicotinic acetylcholine receptor (nAChR) heterologously expressed in Xenopus oocytes. Cy5-RgIA was then used to successfully visualise nAChRs in the RAW264.7 mouse macrophage cell line. This work introduced not only a new and valuable nAChR probe, but also a new versatile synthetic strategy that facilitates production of milligram to gram quantities of fluorophore-labelled peptides at low cost, which is often required for in vivo experiments. The strategy is compatible with Boc- and 9-fluorenylmethoxycarbonyl (Fmoc)-chemistry, allows site-specific labelling of free amines anywhere in the peptide sequence, and can also be used for the introduction of Cy3/Cy5 fluorescence resonance energy transfer (FRET) pairs.