This article describes an optical method based on the association of surface plasmon resonance (SPR) with chitosan (CS) film and its nanocomposites, including zinc oxide (ZnO) or graphene oxide (GO) for glyphosate detection. CS and CS/ZnO or CS/GO thin films were deposited on an Au chip using the spin coating technique. The characterization, morphology, and composition of these films were performed by Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle technique. Sensor preparation conditions including the cross-linking and mobile phase (pH and salinity) were investigated and thoroughly optimized. Results showed that the CS/ZnO thin-film composite provides the highest sensitivity for glyphosate sensing with a low detection limit of 8 nM and with high reproducibility. From the Langmuir-type adsorption model and the effect of ionic strength, the adsorption mechanisms of glyphosate could be controlled by electrostatic and steric interaction with possible formation of 1:1 outer-sphere surface complexes. The selectivity of the optical method was investigated with respect to the sorption of glyphosate metabolite (aminomethylphosphonic acid) (AMPA), glufosinate, and one of the glufonisate metabolites (3-methyl-phosphinico-propionic acid) (MPPA). Results showed that the SPR sensor offers a very good selectivity for glyphosate, but the competition of other molecules could still occur in aqueous systems.