In this work, four novel defective MIL-101(Fe) catalysts with coordinatively unsaturated sites were successfully prepared via a facile synthesis strategy by employing benzoic acid, acetic acid, oxalic acid, or citric acid as a modulator. The modified catalysts were demonstrated the existence of defects in the parent framework by a series of characterizations. As compared to the initial MIL-101(Fe), the electronic structure of defective MIL-101(Fe) catalyst was effectively adjusted; meanwhile, the coordinatively unsaturated Fe sites were efficiently generated and the pore sizes were enlarged. Besides, the defective MIL-101(Fe) catalysts exhibited excellent catalytic performance for rhodamine B degradation by persulfate activation. To be specific, the degradation rates of rhodamine B increased from 58.70 to 94.05%, 86.11%, 78.70%, and 82.62%, respectively. The defective MIL-101(Fe) with coordinatively unsaturated sites showed good reusability and stability, and the probable catalytic mechanism was also investigated.