We report the development of simple fluorogenic probes that report on the activity of both bacterial and mammalian uracil–DNA glycosylase (UDG) enzymes. The probes are built from short, modified single-stranded oligonucleotides containing natural and unnatural bases. The combination of multiple fluorescent pyrene and/or quinacridone nucleobases yields fluorescence at 480 and 540 nm (excitation 340 nm), with large Stokes shifts of 140–200 nm, considerably greater than previous probes. They are strongly quenched by uracil bases incorporated into the sequence, and they yield light-up signals of up to 40-fold, or ratiometric signals with ratio changes of 82-fold, on enzymatic removal of these quenching uracils. We find that the probes are efficient reporters of bacterial UDG, human UNG2, and human SMUG1 enzymes in vitro, yielding complete signals in minutes. Further experiments establish that a probe can be used to image UDG activity by laser confocal microscopy in bacterial cells and in a human cell line, and that signals from a probe signalling UDG activity in human cells can be quantified by flow cytometry. Such probes may prove generally useful both in basic studies of these enzymes and in biomedical applications as well.