Abstract Chiral fluorescent probes based on small organic molecules present a promising platform for advanced bioimaging applications. In this work, we have developed a helically chiral spiro-shaped carbofluorescein with red emission (λem ≤ 613 nm). Configurationally stable enantiomers were separated, and electronic circular dichroism (ECD) and circularly polarized luminescence (CPL) evaluated. Preliminary results showed cellular permeability and intracellular emission.