In the presence of dimethylallyl diphosphate, FgaPT2 was used to catalyze the synthesis of C-4 prenylated indole diketopiperazine. The synthesized product has been tested for anti-tumor, anti-bacterial, anti-fungal, and antioxidant activity. For the product with the highest biological activity, the feasibility of site-directed mutagenesis to increase the yield of enzyme synthesis was studied. The results showed that FgaPT2 enzyme catalyzed the synthesis of 7 C4-prenylated indole diketopiperazines, and FgaPT2 has a certain selectivity for substrates. C4-Prenylation significantly improves the biological activity of indoledikepiperazine, especially the prenylation product 6b. Site-directed mutagenesis of Arg244 showed that 52.6% of FgaPT2 mutants increased the synthesis yield of 6b. Kinetic parameters verify the interaction between 6a and mutant FgaPT2, which can increase the conversion rate of isoprenyl groups.