Objective To develop a method for fast replacement of promoters to improve protein production. Results A method (entitled retreat to advance or "ReToAd"), which includes a deleting PCR and a touchdown PCR, was validated by replacing seven IPTG-inducible promoters with enhanced green fluorescent protein (eGFP). The seven promoters were fully recovered by sequencing only 30 clones. The activity of E. coli harboring omega-transaminase (omega-TA) was increased from 112 U/mg cells (T7 promoter) to 147 U/mg cells (Trc promoter) by combining ReToAd and screening experiments. After screening a library comprising glutamate dehydrogenase (GDH) expressed by different promoters, the activity of E. coli cell harboring Trc-promoter-expressed GDH was similar to 31-fold higher than that of T7-promoter-expressed GDH. Conclusions The "ReToAd" for in situ rapid replacement of promoters was developed and optimized, and one round of "ReToAd" can be completed within 3 days.