One-pot enzymatic production of nigerose was demonstrated from abundantly available sugar resources, including maltose, cellobiose, sucrose and starch. (i) 319 mM nigerose was generated from 500 mM maltose by the combined actions of maltose phosphorylase and nigerose phosphorylase, which share the same β-D-glucose 1-phosphate, in the presence of phosphate. The yield was 62% based on the concentration of maltose as the starting material. (ii) 129 mM nigerose was produced from 250 mM cellobiose by cellobiose phosphorylase and nigerose phosphorylase in the presence of phosphate, in combination with the enzymatic pathway to convert α-D-glucose 1-phosphate to β-D-glucose 1-phosphate via D-glucose 6-phosphate by the combined actions of α-phosphoglucomutase and β-phosphoglucomutase, resulting in a yield of 52%. (iii) 350 mM nigerose was produced from 500 mM sucrose by substituting cellobiose phosphorylase with sucrose phosphorylase and adding xylose isomerase, giving a yield of 67%. (iv) 270 mM nigerose was generated from 100 mg/mL starch and 500 mM D-glucose by the concomitant actions of glycogen phosphorylase, isoamylase, α-phosphoglucomutase, β-phosphoglucomutase and nigerose phosphorylase, in the presence of phosphate. In addition, 280 mM 3- O -α-D-glucopyranosyl-D-galactose was produced by substituting D-glucose with D-galactose. Based on the concentrations of D-glucose and D-galactose as the starting materials, the yields were calculated to be 52 and 56%, respectively. These one-pot enzymatic approaches can be extended to include practical production of a variety of oligosaccharides by substituting nigerose phosphorylase with other β-D-glucose 1-phosphate-forming phosphorylases together with various carbohydrate acceptors.