Glyphosate is widely used in agriculture, and its residues accumulate in soil and water, which adversely affects human health. The existing colorimetric methods for glyphosate detection are usually achieved by inhibiting the catalytic activity of natural enzymes or nanozymes, and often require the participation of easily decomposable H2O2, leading to unstable detection capabilities or even false positive results. This article reports a smartphone-assisted sensor for colorimetric detection of glyphosate in several food samples based on enhancing the laccase-like activity of cubic Ag2O nanoparticles. Glyphosate can significantly enhance the generation of radicals and electron transfer in cubic Ag2O reaction solution, causing an obvious change in the color of the system, which can be easily distinguished by naked eye. The established sensor has excellent detection performance, with limit of detection as low as 2.09 μg/L, and it is highly selective to other competitive substances. The method of enhancing the enzyme-mimic activity of nanomaterials to detect glyphosate residues is proposed for the first time, which can avoid false positive results caused by traditional enzyme inhibition methods and complex food matrices. Moreover, smartphones can simplify the conventional analysis procedure and achieve convenient on-site monitoring of glyphosate pesticides in food.