To improve the growth, yield and quality of Dendrobium huoshanense, Fe ₃O ₄ NPs has been used as nano-fertilizer. Safety of Fe ₃O ₄ NPs in medicinal production on D. huoshanense must be critically evaluated. In this study, two-year-old D. huoshanense was cultured in a solution containing Fe ₃O ₄ NPs (100 or 200 mg/L) for 21 days. The results were investigated by a combination of phenotypic and non-target metabolomics based on gas chromatography-mass spectrometry (GC-MS). Fe ₃O ₄ NPs induced not only earlier flowering and increased sugar content, photosynthesis, but also stress to plants, increased MDA content and related antioxidant enzymes activities. Fe ₃O ₄ NPs caused a significant Fe and some other nutrient elements (Mn, Co, B, Mo) accumulation in stems based in Inductively Coupled Plasma Mass Spectrometry analysis. Metabolomics revealed that the metabolites were reprogrammed in D. huoshanense by Fe ₃O ₄ NPs exposure. Fe ₃O ₄ NPs can inhibit antioxidant defense-related pathways, suggesting that Fe ₃O ₄ NPs had the antioxidant ability with plants accumulating relative metabolites to cope with stresses. As the first study associating Fe ₃O ₄ NPs with the quality of D. huoshanense, it provided vital insights into the molecular mechanisms of how D. huoshanense responds to Fe ₃O ₄ NPs, ensuring the reasonable use of Fe ₃O ₄ NPs as nano-fertilizer.