A greenhouse pot experiment was performed to investigate the enhancement of repeated applications of citric acid (CA), ethylenediamine disuccinic acid (EDDS), and Oxalic acid (OA) on phytoremediation of uranium (U) contaminated soil by Macleaya Cordata. The chelates followed the order CA > EDDS > OA in terms of the enhancement on uranium uptake by M. cordata. The repeated applications of the chelates were found to be more effective than the one time application at the equal dose as the U concentration of soil solution increased significantly from the 8th to 14th day. The repeated applications of 10 mmol kg CA promoted the solubilization of U in the U-contaminated soil by significantly decreasing the pH of soil solution, achieved the maximum U concentration of soil solution (1463.6 µg L), bioconcentration factors (BCFs, 11.4), bioaccumulation factors (BAFs, 21.4) and transfer factors (TFs, 1.9), which were 215.2, 5.7, 30.6 and 16.3 times as compared with the control group, respectively. The three applied chelates significantly affected the activities of the antioxidant enzymes in the leaves. Repeated applications of CA further enhanced the activities of the antioxidant enzymes in the leaves of M. cordata as compared with the control, EDDS and OA, mitigated the oxidative stress induced by uranium and chelates, and maximized the enhancement on the uranium uptake, which will be beneficial for the enhancement on the phytoremediation of uranium contaminated soil by U hyperaccumulating plants. These results indicated that the phytoavailability of uranium in soil solution as well as the accumulation of U by M. cordata were both significantly increased after repeated applications of CA, and that the repeated applications of 10 mmol kg CA increased the activities of the antioxidant enzymes and promoted U accumulation by M. cordata. The study provided an environmentally friendly alternative for the enhancement on the phytoremediation of uranium contaminated soil using M. cordata.