This study aimed to assess the uptake of diclofenac, a widely used nonsteroidal anti-inflammatory pharmaceutical, by a macrophyte Cyperus alternifolius in a mesocosm-scale free water surface (FWS) constructed wetland. Quantitative analysis of diclofenac concentrations in water solution and plant tissues was conducted by high performance liquid chromatography analysis after sample pre-treatment with solid-phase extraction and liquid extraction, respectively. The FWS with Cyperus alternifolius obtained a maximum 69.3% diclofenac removal efficiency, while a control system without plant only had a removal efficiency of 2.7% at the end of the experiment period of 70 days. Based on mass balance study of the experimental system, it was estimated that plant uptake and in-plant conversion of diclofenac contributed about 21.4% of the total diclofenac removal in the mesocosm while the remaining 78.6% diclofenac was eliminated through biotic and abiotic conversion of diclofenac in the water phase. Diclofenac on the root surface and in roots, stems and leaves of Cyperus alternifolius was found at the concentrations of 0.15–2.59 μg/g, 0.21–2.66 μg/g, 0.06–0.53 μg/g, and 0.005–0.02 μg/g of fresh weight of plant tissues, respectively. The maximum bioaccumulation factor of diclofenac was calculated in roots (21.04) followed by root surface (20.49), stems (4.19), and leaves (0.16), respectively. Diclofenac translocation potentiality from root to stem was found below 0.5, suggesting a slow and passive translocation process of diclofenac. Current study demonstrated high potential of Cyperus alternifolius for phytoremediation of diclofenac in FWS and can be applied in other engineered ecosystems.