Thioredoxin reductase(TrxR)is a selenium containing protein which play important roles in drug resistance and proliferation of tumor cells.1 The determination of TrxR activity of biological samples is hindered by the high concentration of small thiol molecules.Here,we report a TrxR – responding OFF-ON fluorescent probe to visualize TrxR activity.A α,β-unsaturated ketone moiety is coupled with a fluorescent group and act as a fluorescenc quencher,covalent modification of active site Cys497 and Sec498 residues in the C terminal by the α,β-unsaturated ketone moiety switch on the fluorescent probe.