Pickering emulsion systems serve as an advanced platform for efficient fine chemical production in biphasic enzymatic catalysis, though their applications are currently limited to a few commercial enzyme classes. Herein, we designed an anchor peptide-based immobilization strategy for Pickering emulsions to achieve efficient glycosylation of natural products by glycosyltransferases (GTs). Firstly, through enzyme mining, natural GTs were utilized to synthesize pharmaceutically important acacetin glucoside and galactoside. However, the best-performing enzymes, BacGT and BarGT-3, still showed low conversions for acacetin glucoside (<
40%) and galactoside (<
10%). Then, Spy chemistry was employed to cyclize these two GTs (Spy_BacGT and Spy_BarGT-3) for improved robustness, and a Pickering emulsion was formed with the free two Spy_GTs, using 20% 2-hexanone and 1% mesoporous silica nanoparticles (carriers of mesoporous, CM), achieving 70% and 66% conversions of acacetin glucoside and galactoside, respectively. Further immobilization of the cyclized GTs onto CM via the anchor peptide liquid chromatography peak I (LCI) (Spy_BacGT/BarGT-3_LCI@CM) further enabled the system to reach more than 90% conversions of acacetin glycosides, retaining 90% conversions after 6-8 cycles. Moreover, this strategy was applied to GTs exhibiting substrate selectivity, achieving efficient nonselective catalysis. This study provides a simple and efficient immobilization strategy to broaden the applications of the Pickering emulsion system in enzymatic glycosylation.