Abstract A facile, highly sensitive colorimetric strategy for dihydronicotinamide adenine dinucleotide (NADH) detection is proposed based on anti-aggregation of gold nanoparticles (AuNPs) via boronic acid–diol binding chemistry. The aggregation agent, 4-mercaptophenylboronic acid (MPBA), has specific affinity for AuNPs through Au–S interaction, leading to the aggregation of AuNPs by self-dehydration condensation at a certain concentration, which is responsible for a visible color change of AuNPs from wine red to blue. With the addition of NADH, MPBA would prefer reacting with NADH to form stable borate ester via boronic acid–diol binding dependent on the pH and solvent, revealing an obvious color change from blue to red with increasing the concentration of NADH. The anti-aggregation effect of NADH on AuNPs was seen by the naked eye and monitored by UV–vis extinction spectra. The linear range of the colorimetric sensor for NADH is from 8.0×10−9M to 8.0×10−6M, with a low detection limit of 2.0nM. The as-established colorimetric strategy opened a new avenue for NADH determination.