Glucose-6-phosphate dehydrogenase (G6PD) is the principal enzyme in the pentose phosphate pathway that plays a fundamental role in the production of nicotinamide adenine dinucleotide phosphate, which is very important in preventing the oxidation of cells, especially red blood cells. This enzyme deficiency was associated with many disorders, the most common of which were hemolysis episodes. In the last decade, nanoparticles have been used to design optical and electronic sensors due to their unique properties. This report presents a new colorimetric method that used silver nanoparticles to detect glucose 6-phosphate dehydrogenase activity directly. The glucose-6-phosphate dehydrogenase detection mechanism was based on an aggregation of silver nanoparticles, leading to increased nanoparticle size, which causes discoloration. In the presence of the enzyme, the color of the solution was yellow, and when the enzyme was not present, the color of the solution was grayish. Utilizing this method, colorimetric sensing of glucose 6-phosphate dehydrogenase was gained with a detection limit of 0.009 U ml−1 and a linear range of 0–16.0 U ml−1. In this way, the presence or absence of the enzyme can be easily detected with the naked eye during one step.