SummaryDissolved oxygen (DO) is an important influencing factor in the process of aerobic microbial fermentation. Spinosad is an aerobic microbial‐derived secondary metabolite. In our study, spinosad was used as an example to establish a DO strategy by multi‐scale analysis, which included a reactor, cell and gene scales. We changed DO conditions that are related to the characteristics of cell metabolism (glucose consumption rate, biomass accumulation and spinosad production). Consequently, cell growth was promoted by maintaining DO at 40% in the first 24 h and subsequently increasing DO to 50% in 24 h to 96 h. In an in‐depth analysis of the key enzyme genes (gtt, spn A, spn K and spn O), expression of spinosad and specific Adenosine Triphosphate (ATP), the spinosad yield was increased by regulating DO to 30% within 96 h to 192 h and then changing it to 25% in 192 h to 240 h. Under the four‐phase DO strategy, spinosad yield increased by 652.1%, 326.1%, 546.8%, and 781.4% compared with the yield obtained under constant DO control at 50%, 40%, 30%, and 20% respectively. The proposed method provides a novel way to develop a precise DO strategy for fermentation.