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An optical sensor based on pyranine immobilized on aminopropyl-modified mesoporous silica films was developed for paraquat detection in aqueous solutions. An electrochemically assisted self-assembly method was used to deposit mesoporous silica film on fluorine-doped tin oxide glass. The obtained films were modified with various concentrations of 3-aminopropyl triethoxysilane (APTES) before the immobilization of pyranine. Cyclic voltammetry, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy were used to characterize the films. Pyranine-immobilized films gave an emission at 506 nm with an excitation at 450 nm. The fluorescence signal was quenched in the presence of paraquat. The films modified with 3% APTES provided the optimum response to paraquat. The developed films had a linear response to paraquat in the concentration range of 1 to 10 ppm at the optimum conditions, with a detection limit of 0.80 ppm. The developed method was used to quantify paraquat in sugarcane peel and tap water samples with satisfactory results.