Invertase (α-glucosidase) is one of the most important honey enzymes; it hydrolyses sucrose into fructose and glucose during honey ripening process. Next to the basic honey ingredients (glucose, fructose, water), invertase activity is one of the main characterising parameter of honey: it can be used as indicator of aging and/or overheating, but it also may give information about adulteration. Our aim was to develop a novel analytical method for the fast determination of invertase activity that can be used during quality control of honey samples. Our assay based on the application of an artificial substrate, namely p-nitrophenyl-α-D-glucopyranoside. p-Nitrophenol produced by the enzyme reaction is detected by amperometric method which is much more sensitive than the traditional spectrophotometric determination. Screen-printed carbon electrodes and a potentiostat were used for amperometric measurement. Our measuring system worked in flow injection system. The measuring parameters (polarization potential, pH etc.) were optimized. The applicability of the method was tested for detection of α-glucosidase enzyme activity.