Actual biomass of microalgae was tested as a fermentation substrate for microbial production of 2-pyrone 4,6-dicarboxylic acid (PDC). Acid-hydrolyzed green microalgae Chlorella emersonii (algae hydrolysate) was diluted to adjust the glucose concentration to 2 g/L and supplemented with the nutrients of Luria–Bertani (LB) medium (tryptone 10 g/L and yeast extract 5 g/L). When the algae hydrolysate was used as a fermentation source for recombinant Escherichia coli producing PDC, 0.43 g/L PDC was produced with a yield of 20.1% (mol PDC/mol glucose), whereas 0.19 g/L PDC was produced with a yield of 8.6% when LB medium supplemented with glucose was used. To evaluate the potential of algae hydrolysate alone as a fermentation medium for E. coli growth and PDC production, the nutrients of LB medium were reduced from the algae hydrolysate medium. Interestingly, 0.17 g/L PDC was produced even without additional nutrient, which was comparable to the case using pure glucose medium with nutrients of LB medium. When using a high concentration of hydrolysate without additional nutrients, 1.22 g/L PDC was produced after a 24-h cultivation with the yield of 16.1%. Overall, C. emersonii has high potential as cost-effective fermentation substrate for the microbial production of PDC.