The sandwich assay was used to construct a test strip based on the colloidal gold labeled aptamer. The purpose was to develop a practical As(III) detection application device. Through the specific combination of As(III) and the gold labeled aptamer, As(III) would be intercepted at the test line of the test strip. In this study, nano-gold was used as color developing material, and its particle diameter was (13 ± 1) nm. After optimizing the reaction conditions, Satorius CN 140 was selected as the nitrocellulose membrane. The optimal ratio of capture probe streptoavidin and biotin on the control line was 1∶4, and the corresponding concentration of scribing was 10 μmol/L. The optimal ratio of NHS and EDC on the test line was 1∶3, and the optimal concentration of gold standard aptamer was 0.5 μmol/L. The color developing result was observed under the optimal reaction conditions, and the color response signal formed a good gradient relationship with the concentration of As(III). The detection limit was 0.1 μmol/L. This test strip had high specificity for As(III) and good stability. Its detection cost was less than 1 yuan, which could meet the requirements of real-time detection of As(III) for the general population in low-income areas.