The fluorescence-based assay of iodide ion (I−) has been extensively studied by the use of different sensing probes and techniques, but it remains a tricky task to eliminate the interference of chloride ion (Cl−) for the analysis of low-level I− in complex genuine samples. Herein, we develop a redox pretreatment strategy for specific separating I− from human urine. Simultaneously, a novel ratiometric fluorescent probe is constructed by a simple mixing of dimer DNA silver nanoclusters (dDNA-AgNCs) and carbon dots (CDs) with the ratio of 5:1 in fluorescent intensity, and used for visual assay of I−. After addition of I−, the fluorescence of orange dDNA-AgNCs can be quenched by I− as the result of I−-induced oxidative etching and aggregation of dDNA-AgNCs, while blue CDs as the stable internal standard are unresponsive to I−. With the increase of I−, the fluorescence intensity ratio (I577/I446) of binary-color probe gradually decreased, which leads to color variation from salmon pink to lighter salmon pink to lilac to light steel blue to final deep sky blue (under a UV lamp) with a sensitive detection limit of 19.8 nM. The assay for I− can also be convenient to implement for visual monitoring of I− by observing color change of test paper printed with the ratiometric probe, responding to 50 nM that is about 1 order of magnitude lower than the median urinary I− concentration defined by the World Health Organization (WHO) for school-age children. The sensitive test paper can provide an advanced platform for colorimetric and visual monitoring of I− in human urine.