We developed a label-free and washing-free method for biomolecular detection using a personal glucose meter (PGM). ATP was selected as a model target, and cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were adopted to link the amount of ATP to glucose that is detectable by a hand-held PGM. In principle, the presence of target ATP enables hexokinase to catalyze the conversion of glucose to glucose 6-phosphate by providing a phosphate group to glucose, and thus the amount of glucose is decreased in proportion to the amount of ATP. In addition, adenosine 5'-diphosphate (ADP), which is generated after hexokinase-catalyzed enzymatic reaction, is recovered to ATP by a pyruvate kinase enzyme. The regenerated ATP is again supplemented to catalyze multiple rounds of cascade enzymatic reactions, leading to signal amplification. As a result, the change of glucose amount that is inversely proportional to ATP amount is simply measured by a hand-held PGM. By employing this strategy, we successfully determined ATP down to 49 nM with high selectivity even in real samples such as tap water, human serum, and bovine urine. Importantly, the developed system does not require expensive modification and washing steps but is conveniently operated with a commercially available PGM, which would pave the way for the development of a simple and cost-effective sensing platform.