The superb specificity and potency of biological toxins targeting various ion channels and receptors are of major interest for the delivery of therapeutics to distinct cell types and subcellular compartments. Fused with reporter proteins or labelled with fluorophores and nanocomposites, animal toxins and their detoxified variants also offer expanding opportunities for visualisation of a range of molecular processes and functions in preclinical models, as well as clinical studies. This article presents state-of-the-art optical probes derived from neurotoxins targeting ion channels, with discussions of their applications in basic and translational biomedical research. It describes the design and production of probes and reviews their applications with advantages and limitations, with prospects for future improvements. Given the advances in imaging tools and expanding research areas benefiting from the use of optical probes, described here resources should assist the discovery process and facilitate high-precision interrogation and therapeutic interventions.