Conventional pesticide residue detection still suffers from numerous steps, is long time-consuming, and insufficient delicacy. According to ninhydrin colouring and the principle of localized surface plasmon resonance (LSPR) enhancing absorption, glyphosate in water samples can be detected by ultraviolet-visible spectroscopy (UV-Vis). Furthermore, density functional theory is used to analyze the enhancement mechanism of absorption of purple color dye (PD) products. The PD product displays a maximum absorption of around 570 nm when glyphosate reacts with ninhydrin is detected by UV-Vis. There are slight shifts from 570 nm in the UV-Vis spectrum to 568 nm with a stronger peak when the PD product is absorbed on Ag NPs, and the limit of detection at 2.017 4×10-11 mol·L-1, which is much lower than 6.5×10-7 mol·L-1 limit of detection reported. Gaussian 09 software carried out that the PD product would attach to Ag NPs via an Ag-O bond through ninhydrin’s CO group vertically. MEP mapping provides CO group interaction with Ag NPs stable, CO group coupled C-N comprise a large π-conjugated system in their plane. The color of the C-N group is blue, which suggests that C-N coupled with the CO group are the chromophore in the PD product. Thus, an indirect detection method derived from ninhydrin can be used for glyphosate detection in water samples. The LSPR effect of Ag NPs enhances the absorption intensity with higher sensitivity than the conventional method.