A simple and selective assay for detecting methylglyoxal (MGO), a metabolite associated with diabetes, was developed by combining a bio-inspired chemical reaction with the anti-aggregation of gold nanoparticles. This assay could detect MGO at as low as 1 μM by the naked eye and 0.05 μM by UV/vis spectrometry, within the clinical range marking oxidative stress in diabetes, and demonstrated high selectivity over other physiologically relevant ketones and aldehydes.