A fluorescent sensor L based on a pyrazolopyrimidine core simultaneously detects Cu and Ni ions by photoluminescence quenching, even in the presence of other metal cations. Sensor L possesses high association constants of 5.24 × 10 M and 2.85 × 10 M and low detection limits of 0.043 μM and 0.038 μM for Cu and Ni, respectively. The binding stoichiometry ratios of L to Cu or Ni is 1:1 as determined by Benesi-Hildebrand and Job's plots, and by crystal structures. DFT calculations on L-Cu indicated reduced electron donation from the coordinated pyrazolopyridine to the fused pyrimidine and pendant phenyl group which, together with a smaller HOMO-LUMO orbital gap could favour non-radiative decay and explain the observed fluorescence quenching. Sensor L possessed low cytotoxicity and good imaging characteristics for Cu and Ni in living cells, suggesting potential applications for detecting Cu and Niin vivo.