Biosynthesis involving multi-enzymatic reactions is usually an efficient and economic method to produce plentiful important molecules. To increase the product yield in biosynthesis, the involved enzymes can be immobilized to carriers for enhancing enzyme stability, increasing synthesis efficiency and improving enzyme recyclability. Hydrogels with three-dimensional porous structures and versatile functional groups are promising carriers for enzyme immobilization. Herein, we review the recent advances of the hydrogel-based multi-enzymatic system for biosynthesis. First, we introduce the strategies of enzyme immobilization in hydrogel, including the pros and cons of the strategies. Then we overview the recent applications of the multi-enzymatic system for biosynthesis, including cell-free protein synthesis (CFPS) and non-protein synthesis, especially high value-added molecules. In the last section, we discuss the future perspective of the hydrogel-based multi-enzymatic system for biosynthesis.