The presence of cadmium ions (Cd2+) in environmental samples demands a fast, sensitive, and selective analytical method that can measure toxic levels. Biosensors based on aptamers (aptasensors) have been developed, but some of them suffer from poor sensitivity and specificity due to the immobilization of aptamers. Here, we employed circular dichroism, molecular docking, and molecular dynamics simulation to reveal that the aptamer gradually undergoes significant conformational changes upon Cd2+ binding. This fact highlights the advantages of biosensors based on free aptamers. So, keeping these results, an analytical method was established for the detection of Cd2+ by utilizing capillary zone electrophoresis (CZE), which is adapted for the free aptamer. So, CZE equipped with aptamer as a detection probe can detect Cd2+ within 4 min in the range from 5 to 250 nM with R2 = 0.994, limit of detection 5 nM (signal-to-noise ratio = 3), and recovery from 92.6 ± 1.6 to 107.4 ± 1.0% in river water samples. Furthermore, the detected concentration in water samples is below the harmful levels (267 nM) recommended by World Health Organization standards in drinking water. This method displays a high sensitivity and specificity for Cd2+. It is found to be superior to existing methods, which use immobilized aptamers, and can be readily expanded to design aptasensors for other targets.