Pentacyclic triterpenoids have wide applications in the pharmaceutical industry. The precise glucosylation at C‐3 OH of pentacyclic triterpenoids mediated by uridine 5'‐diphospho‐glucosyltransferase (UDP‐glucosyltransferase UGT) is an important way to produce valuable derivatives with various improved functions. However, most reported UGTs suffer from low regiospecificity toward the OH and COOH groups of pentacyclic triterpenoids, which significantly decreases the reaction efficiency. Here, two new UGTs (UGT73C33 and UGT73F24) were discovered in Glycyrrhiza uralensis. UGT73C33 showed high activity but poor regioselectivity toward the C‐3 OH and C‐30 COOH of pentacyclic triterpenoid, producing three glucosides. UGT73F24 showed rigid regioselectivity toward C‐3 OH of typical pentacyclic triterpenoids producing only C‐3 O‐glucosylated derivatives. In addition, UGT73C33 and UGT73F24 showed a broad substrate scope toward typical flavonoids with various sugar donors. Next, the substrate recognition mechanism of UGT73F24 toward glycyrrhetinic acid (GA) and UDP‐glucose was investigated. Two key residues, I23 and L84, were identified to determine activity, and site‐directed mutagenesis of UGT73F24‐I23G/L84N increased the activity by 4.1‐fold. Furthermore, three in vitro GA glycosylation systems with UDP‐recycling were constructed, and high yields of GA‐3‐O‐Glc (1.25 mM), GA‐30‐O‐Glc (0.61 mM), and GA‐di‐Glc (0.26 mM) were obtained. The de novo biosynthesis of GA‐3‐O‐glucose (26.31 mg/L) was also obtained in engineered yeast. This work discovered two new UDP‐glucosyltransferases from Glycyrrhiza uralensis. UGT73C33 showed low regioselectivity toward C‐3 OH and C‐30 COOH of typical pentacyclic triterpenoids, but UGT73F24 showed rigid regioselectivity toward C‐3 OH of typical pentacyclic triterpenoids. Meanwhile, the critical residues responsible for determining activity of UGT73F24 were identified, which helped understand the molecular basis of UGT73F subfamily members. With UGT73F24 and its variant with improved activity, both in vitro and in vivo controllable glycosylation systems for pentacyclic triterpenoids were established.