Glycolaldehyde (GA), the smallest sugar, has significant potential as a biomass-derived platform chemical and is a key metabolite in several synthetic pathways for one-carbon metabolism and new-to-nature photorespiration. This study introduces two metabolic schemes for engineering Escherichia coli into GA biosensors. Through creating GA-dependent auxotrophies, we link growth of these strains to GA-dependent biosynthesis of the essential vitamin pyridoxal-5-phosphate, and 2-ketoglutarate, respectively. We characterized and optimized these strains for the quantification of externally added GA from 2 µM to 1.5 mM. We also demonstrate the capability of these strains to detect GA that is produced intracellularly through different metabolic routes and from different substrates such as xylose, ethylene glycol, and glycolate. Our biosensors offer complementary sensitivities and features, opening up different applications in metabolic engineering and synthetic biology, which we demonstrate in a proof-of-principle by providing the first in vivo demonstration of the reduction of glycolate to GA by a new-to-nature route using engineered enzymes.