The function of membrane-localized sodium iodide symporter (NIS) determines the efficacy of radioiodine therapy in thyroid cancer. Here, we describe a dual mode reporter fused with human NIS (hNIS) and a red fluorescent protein named tandem dimeric Tomato (tdTomato) for the in vitro and in vivo imaging of hNIS protein expression, localization, and iodide uptake function. Human cervical epithelial adenocarcinoma cell line (HeLa)-hNIS/tdTomato cells were established by transducing a fusion gene expressing hNIS/tdTomato under the control of a cytomegalovirus promoter. Fluorescence imaging, confocal microscopy, and an 125I uptake assay were performed to validate the integrity of the fusion protein. Actinomycin D and cycloheximide were used to block newly synthesized hNIS proteins. In vivo images were acquired using a gamma camera and a Maestro fluorescence imaging device. The fluorescence intensity of membrane-localized hNIS and 125I uptake both were increased after heat shock. Scintigraphy and fluorescence imaging indicated specific accumulation of the hNIS/tdTomato fusion protein in xenografted tumors, supporting the utility of this system for in vivo monitoring of hNIS expression and activity. We developed a novel hNIS/tdTomato dual mode reporter that enables visualization of the expression, localization, and iodine uptake function of hNIS in vitro and in vivo.