Monitoring the levels of pesticides on plant tissues is important for achieving effective protection of crops after application, as well as ensuring low levels of residues during harvest. In this study, a simple, rapid, and fieldable colorimetric method for detecting the pesticide glyphosate (Gly) on the plant tissues in situ using cysteamine-modified gold nanoparticles (AuNPs-Cys) has been developed. The aggregation of AuNPs-Cys in the presence of Gly results in a consequent color change from red to blue (or purple), which could be observed visually on the surface of plant tissues. With the naked eye, we successfully detected Gly spiked on the surface of spinach, apple, and corn leaves in situ. Further verification and quantification were achieved using surface-enhanced Raman spectroscopy (SERS) which uses AuNPs-Cys as the substrate. Moreover, application of this method was demonstrated through the evaluation of the Gly distribution on plant tissues which could greatly facilitate the development of precision agriculture technology.