Abstract: Phytoremediation is an environmentally friendly and highly promising method for soil remediation, with the core issue being the selection of suitable plants and applicable conditions. During the remediation of uranium-contaminated environments, the enrichment of uranium adversely affects the normal growth of plants, causing stress effects. This study aimed to explore the stress response mechanism of Bidens pilosa L. to uranium in the soil and the influence of chelating agents on its uranium enrichment capacity through experiments on the remediation of uranium-contaminated soil. The phenotypic, physiological, and chlorophyll fluorescence parameters of Bidens pilosa L., as well as its uranium enrichment capacity, were studied under the influence of chelating agents and uranium. The results show that with the increase of uranium concentration, the levels of soluble protein, malondialdehyde (MDA), peroxidase (POD), and catalase (CAT) in Bidens pilosa L. first increase and then decrease. The maximum quantum yield (Fv/Fm) and non-photochemical quenching coefficient (NPQ) significantly decrease with the increase of uranium concentration, while the actual quantum yield (ΦPSⅡ) gradually increases. The application of chelating agents significantly enhances the uranium enrichment capacity of Bidens pilosa L. Low concentrations of chelating agents promote the synthesis of soluble protein, POD, and CAT, and reduce MDA content. Additionally, low concentrations of chelating agents increase the Fv/Fm and ΦPSⅡ values of Bidens pilosa L. However, they could also affect the open center structure of PSⅡ in plants, leading to the damage of the photoprotection system. High concentrations of chelating agents could directly cause plant death. In terms of enrichment capacity, when the soil uranium concentration is 50 mg/kg, under the action of 20 mmol/kg oxalic acid, Bidens pilosa L. achieves a maximum total enrichment coefficient of 6.29, with an above-ground part enrichment coefficient of 4.26 and a transfer coefficient of 2.09, exceeding the lower limits defined for hyperaccumulator plants. Thus, it can be defined as a hyperaccumulator plant, but under these conditions, Bidens pilosa L. does not survive for more than 24 h. Therefore, if Bidens pilosa L. is used as a phytoremediation material for uranium-contaminated soil, the application of 10 mmol/kg citric acid can enhance its uranium enrichment capacity while ensuring better survival rates. The above results indicate that by adjusting the concentration of chelating agents, it is possible to effectively increase the uranium enrichment efficiency of Bidens pilosa L. while minimizing damage to the plant itself, thereby playing a more significant role in the remediation of uranium-contaminated soils. Furthermore, understanding the precise biochemical and physiological responses of Bidens pilosa L. to uranium stress and chelating agents can help in developing more refined strategies for using this plant in various contaminated environments. This deeper insight into the plant’s response mechanisms also opens up avenues for interventions to enhance its resilience and accumulation capacity, making phytoremediation a more viable and efficient option for large-scale environmental cleanup efforts.