l-Arginine (Arg) is a popular ingredient in many sport supplements due to its potential benefits for athletes, but taking high doses of Arg causes health problems. Here, a new colorimetric apta-nanosensor is developed using unmodified gold nanoparticles (AuNPs) and aptamer for sensitive and specific Arg detection. In the presence of Arg, the surface protective effect of aptamers on AuNPs is lost, and removing aptamers from the surface of AuNPs leads to the aggregation of AuNPs with NaCl. The color changed from red to blue after aggregation was measured by naked-eye or UV–vis spectrophotometer. The linear dynamic range of 1–180 μM with LOD of 0.25 μM was obtained. This apta-nanosensor can detect Arg on-site and rapidly (about 5 min). The applicability of the designed colorimetric apta-nanosensor was evaluated on sports supplement samples containing a certain amount of Arg. It provided an acceptable response with an average RSD of 5.85 %. The results show that this platform can be a strong and promising method for the detection of other substances used in sports supplements.