A model predictive control (MPC) method was investigated as a route to optimize and control the growth of E. coli BL21 (DE3) and biosynthesis of two different recombinant proteins (nerve growth factor NGF and coat protein of bacteriophage Qβ (Qβ-CP)). To determine the target trajectory for the E. coli cultivation process and estimate the model parameters, the off-line run-to-run optimiza-tion method was used. The proven method allowed us to successfully control the growth of micro-bial biomass, with a deviation of 6–12% from the target trajectory. It was proven that it is possible to obtain a “Golden Batch” profile for the implementation of MPC using datasets from only four to eight fermentation runs. The method showed its robustness when the cultivation of E. coli was carried out with two different titrant supply control systems—volumetric and gravimetric. Fur-thermore, the MPC method exhibited high adaptability, reliability, and resistance to various types of disturbances. MPC proved to be a reliable and effective method for controlling the cultivation and recombinant protein biosynthesis of fast-growing microorganisms such as E. coli.