Uridine 5'-diphosphate-glucuronic acid (UDP-GlcA) and UDP-galacturonic acid (UDP-GalA), the unique carboxylic acid-formed sugar nucleotides, are key precursors involved in the biosynthesis of numerous cell components. Limited availability of those components has been hindering the development of efficient ways towards facile synthesis of bioactive glycans such as glycosaminoglycans. In current study, we biochemically characterized two UDP-sugar pyrophosphorylases from Arabidopsis thaliana (AtUSP) and Bifidobacterium infantis ATCC15697 (BiUSP), and compared their activities towards a panel of sugar-1-phosphates and derivatives. Both enzymes showed significant pyrophosphorylation activities towards GlcA-1-phosphate, and AtUSP also exhibited comparable activity towards GalA-1-phosphate. By combining with monosaccharide-1-phosphate kinases, we have developed an efficient and facile one-pot three-enzyme approach to quickly obtain hundreds milligrams of UDP-GlcA and UDP-GalA.