Thioredoxins (Trxs) and glutaredoxins (Grxs) are the two major thiol-dependent reductases, participating in many important cellular events such as defense against oxidative stress, DNA synthesis and repair. Both Trxs and Grxs have diverse disulfide-containing substrates in the cells to exert their activities, with overlapping functions. Specific methods for measuring the intracellular overall activities of Trxs and Grxs are still lacking. Here we find that TRFS-green, a disulfide containing fluorescent probe which was used to detect thioredoxin reductase (TrxR) in mammalian cells, is a substrate of bacterial Trxs and Grxs, but not a substrate of bacterial TrxR and GSH. This property made TRFS-green work as a probe to measure the overall activities of Trxs and Grxs in bacterial cells. Using various E. coli Trx or Grx null mutant strains, the contribution of different Trxs and Grxs to cellular redox regulation has been clarified, judged by the reducibility towards TRFS-green. E. coli Grx2 and Grx3 unexpectedly exhibited higher activity in reducing the disulfide probe than the other redoxins. In addition, the bacterial disulfide reductase activity was detected to be affected in the ofloxacin bactericidal process. These results show that TRFS-green may be a useful tool for investigating bacterial redox regulation and demonstrating the critical role of E. coli Grxs in maintaining the bacterial intracellular redox balance.