Nickel (Ni) is one of the most toxic metals in human health. Its bioaccumulation in gluten-free crops limits the progressing demand of safe foods for allergic people to gluten. Nanoparticles have shown promising results in enhancing the crop yield and reducing the risk of heavy metal uptake. However, their nanotoxicity has been raised environmental concerns. This study investigated the environmental behavior of Ni (II) with the co-presence of Zinc Oxide Nanoparticles (ZnO-NPs) in sorghum bicolor. The plants were exposed to different treatments of Ni, ZnO-NPs, or their coexistence. The uptake experiments were carried out within nine treatments consisting of 1 or 5 ppm Ni alone or in coexistence with 50 or 100 ppm ZnO-NPs. The physiological impacts on plants as potential fingerprints for nanotoxicity were recorded and assessed in a phenotypic spectrum. The total Ni or Zn contents were quantified using atomic absorption. NPs significantly altered the bioavailability of Ni. The results revealed that at 5 ppm Ni contamination, 50 and 100 ZnO-NPs significantly reduced the Ni uptake by ∼43% and 47%, respectively. Further, the results showed at 50 ppm NPs, the phytotoxicity effects of both Ni and NPs may reduce, leading to higher plant dry biomass yield.Novelty statement Characterization of zinc oxide nanotoxicity threshold by developing a phenotypic spectrum. Also, the study revealed the phytoremediation potential of ZnO nanoparticle in mitigating the nickel uptake in a gluten-free crop (sorghum bicolor).