Objectives A redox potential-driven fermentation, maintaining dissolved oxygen at a prescribed level while simultaneously monitoring the changes of fermentation redox potential, was developed to guide the cultivation progress of recombinant protein expression. Results A recombinantE. coliharboring prolinase-expressing plasmid (pKK-PepR2) was cultivated using the developed process. Two distinct ORP valleys were noticeable based on recorded profile. The first ORP valley is equivalent to the timing for the addition of inducing agent, and the second ORP valley serves to guide the timing for cell harvesting. The final prolinase activity is 0.172 mu mol/mg/min as compared to that of 0.154 mu mol/mg/min where the optical density was employed to guide the timing of inducer addition and an empirically determined length of the cultivation. Conclusion The developed process can be further modified to become an automatic operation.