A C2 symmetric azo derivative of luminol (AzoLum) was used for the selective and specific sensing of Hg2+ using three different modes, ‘cis-trans’ isomerisation, fluorescence quenching and chemiluminescence enhancement. Hg2+ addition to AzoLum triggered the formation of ‘cis’ -AzoLum from the mixture of ‘cis’ and ‘trans’ isomers, alongside the fluorescence quenching. Ratiometric sensing of Hg2+ in the concentration range from 1 ppb to 1000 ppb was studied by following ‘cis-trans’ isomerisation and fluorescence quenching using absorption and fluorescence spectroscopic techniques respectively. On adding cysteine, ‘Cis’-AzoLum- Hg2+ reverts back to a mixture of ‘cis’ and ‘trans’ isomers with the regained fluorescence. The nature of a complex formed between AzoLum and Hg2+ was studied using IR spectroscopy, XRD, TGA and theoretical DFT calculations. With the characteristic phthalhydrazide groups of luminol, AzoLum was also found to sense Hg2+ ratiometrically using chemiluminescence measurements in the presence of hydrogen peroxide. Furthermore, AzoLum was successfully used for bioimaging of Hg2+ in live cells. © 2018 Elsevier B.V.