As a potent herbicide capable of contaminating water and soil environments, paraquat, which is still widely used worldwide, is toxic to mammals, algae, aquatic animals, etc. Paraquat was loaded on novel nanoparticles composed of pectin, chitosan, and sodium tripolyphosphate (PEC/CS/TPP). The size, polydispersity index, and ζ potential of nanoparticles were characterized. Further assessments were carried out by SEM, AFM, FT-IR, and DSC. The encapsulation was highly efficient, and there was a delayed release pattern of paraquat. The encapsulated herbicide was less toxic to alveolar and mouth cell lines. Moreover, the mutagenicity of the formulation was significantly lower than those of pure or commercial forms of paraquat in a Salmonella typhimurium strain model. The soil sorption of paraquat and the deep soil penetration of the nanoparticle-associated herbicide were also decreased. The herbicidal activity of paraquat for maize or mustard was not only preserved but also enhanced after encapsulation. It was concluded that paraquat encapsulation with PEC/CS/TPP nanoparticles is highly efficient and the formulation has significant herbicide activity. It is less toxic to human environment and cells, as was evidenced by less soil sorption, cytotoxicity, and mutagenicity. Hence, paraquat-loaded PEC/CS/TPP nanoparticles have potential advantages for future use in agriculture.