A quinoline-based Schiff base sensor, 6-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxaldehyde-4(N)-phenylsemicarbazone (6MPS), has been developed for selective sensing of methionine and aspartic acid in aqueous medium through “on–off–on” type selective detection of copper ion. Fluorescence imaging of 6MPS, 6MPSC, 6MPSCN, 6MPSC-met, 6MPSCN-met, 6MPSC-asp and 6MPSCN-asp has been successfully demonstrated, in which the sensing probes 6MPSC-met, 6MPSCN-met, 6MPSC-asp and 6MPSCN-asp displayed bright green fluorescence in both in vitro and in vivo live cells.