A novel redox mediator, thiol-reactive phenazine ethosulfate (trPES), was used to modify an enzyme for the first time for biosensor development. Aerococcus viridans-lactate oxidase (LOx), widely used to study lactate biosensors, was modified with a single trPES molecule. A cysteine mutation was introduced into the vicinity of the LOx cofactor to enable the modification by trPES. LOx cysteine mutants were then successfully modified using trPES, thus acquiring quasi-direct electron transfer ability. An electrode immobilized with the trPES-LOx A96L/S210C mutant showed the highest amperometric response currents among the modified LOx cysteine mutants, indicating efficient electron transfer. The position around residues S210 to N212 on the LOx surface (distance <14 Å from FMN N5) is important for the mediator to access the reduced flavin. Then, the performances of the lactate sensor were improved by utilizing LOx A96L/S210C modified with trPES and arPES, another redox mediator used to modify a lysine residue. The lactate sensor has a detection range of up to 1 mM, a sensitivity of 6.62 µA/mM.cm2, and a limit of detection of 9.9 µM. Furthermore, Aspergillus flavus-derived FAD glucose dehydrogenase was successfully modified with trPES and the response currents were obtained, showing the versatility of trPES for modifying other oxidoreductases.