Deoxythymidine diphosphate (dTDP)-activated sugar nucleotides are the most diverse sugar nucleotides in nature. They serve as the glycosylation donors of glycosyltransferases to produce various carbohydrate structures in living organisms. However, most of the dTDP-sugars are difficult to obtain due to synthetic difficulties. The limited availability of dTDP-sugars has hindered the progress in investigating the biosynthesis of carbohydrates and exploring new glycosyltransferases in nature. Here, based on the de novo and salvage biosynthetic pathways, a variety of dTDP-activated sugar nucleotides were successfully prepared in high yields and large scale from the readily available starting materials. The produced sugar nucleotides could provide effective tools for fundamental research in glycoscience.