Lycopene plays a crucial role in the biosynthesis pathway of 2-methyl-derythritol-4-phosphate (MEP) and mevalonic acid (MVA). It is a representative product of isoprenoid family, and a typical product of multi-enzyme catalytic reaction in organism. In this paper, we first introduced the general regulation methods in multi-enzyme synthesis reaction, including the construction of multi gene co-expression plasmid, gene order regulation, promoter and ribosome binding site regulation, gene knockout and replacement, aiming at the optimization strategies of multi-enzyme catalytic reaction in lycopene synthesis pathway. Meanwhile, we introduced several new regulation methods in multi-enzyme reaction, including multi-fragment assembly technology, artificial scaffold self-assembly methods and so on. At last, we summarized the application of these multi-enzyme regulation methods in lycopene synthesis. These methods provide a great inspiration and research foundation for the construction of lycopene-producing strains with high yield.