5-Aminolevulinic acid (ALA) is a common precursor for tetrapyrrole compounds in all kinds of organisms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen (DO) shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombinant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase (ALAS) activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 min in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 min. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g.L-1 (72 mmol.L-1), which is the highest yield in the fermentation broth reported up to now.