Pterostilbene (PST), a 3,5- O-methylated derivative of resveratrol (RSV), is a natural potent antioxidant produced by some plants in trace amounts as defense compounds. It exhibits various health-promoting activities, such as anticancer, antiviral, and antimicrobial effects. Large-scale biosynthesis of PST is crucial due to the challenges associated with extracting it from plants. This study aims to develop an efficient method for PST production using an engineered E. coli strain by feeding RSV as an intermediate compound. We introduced a two-step substrate addition strategy combined with immobilized RSV (IMRSV) on macroporous adsorption resin (MAR) to enhance PST production. Five MARs were selected for RSV immobilization, and the substrate additions strategy and fermentation parameters for PST synthesis were optimized. A maximum PST concentration of 402.78 ± 9.20 mg/L was achieved, representing a 239% increase over the control. The PST titer reached 395.20 ± 23.89 mg/L in a 3-L bioreactor. In conclusion, the combination of a two-step substrate adding system and IMRSV is a promising approach for the economical and industrial-scale production of PST.