Abstract We designed an assay for rapid detection of ascorbic acid (AA) with a DNAzyme cleaving its DNA substrate in the presence of Cu2+ and AA. The sensor consists of two DNA strands that form a complex between each other. The 5′-end of the DNAzyme binds the substrate DNA via Watson–Crick bonding and the 3′-end binds through formation of a DNA-triplex via Hoogsteen hydrogen bonding. The substrate DNA was prepared by two different methods. In the first case the nucleic acid was modified with fluorescein/dabcyl FRET pair across the cleavage site. In the second case the nucleic acid modified with fluorescein was immobilised on gold nanoparticles. DNAzyme contains a loop forming a complex with Cu2+ ions. The oxidation of ascorbic acid (AA) with oxygen yields hydrogen peroxide. The latter interacts with Cu2+ to give hydroxyl radicals. They break substrate DNA in close vicinity to the copper/DNA complex to separate fluorescein from gold nanoparticles leading to the increase in fluorescence intensity. Use of substrate DNA modified with the fluorescein/dabcyl couple allowed to measure AA concentration within 3min with the detection limit of 2.5μM. Employment of gold nanoparticles decorated with fluorescein-modified DNA allowed to improve the detection limit of AA quantification by two orders of magnitude due to enhanced cleavage of DNA catalysed by Au clusters. Fructose, sucrose, glucose, urea, and citric acid did not interfere with our assay even at concentration of 1mM. Good selectivity allowed us to apply our rapid and sensitive assays to detection of AA in vitamin C tablets, urine and orange juice.