DNA-binding fluorescent probes are useful tools in cell biology for various applications enabling visualisation and quantification of DNA. Herein, we report on HeliDye1, a novel cationic helicene-like DNA probe, and its versatile usability. HeliDye1 exhibits fluorescence exclusively in the presence of DNA duplexes containing AT base pairs and (contrary to any recent DNA probe) it remains non-fluorescent in the presence of single-stranded DNA and RNA. Favourable chemical properties of HeliDye1 allowed its use in karyotyping and super-resolution microscopy. We present five more applications of HeliDye1. Viscosimetry showed that HeliDye1 is a minor groove binder. HeliDye1 interacts with double-stranded DNA as an induced (P)-enantiomer, which we confirmed by ECD spectroscopy and DFT calculations. ECD spectroscopy suggested the formation of HeliDye1 dimers. MD simulations predicted the structures of (P)-HeliDye1 dimers inside DNA and showed their stability in AT-rich sequences and instability in GC-rich sequences.