Herein, we report for the first time a colorful chromogenic substrate, which displays vivid color responses in the presence of different concentration of analytes. Our investigation reveals that the selective shortening of gold nanorods (AuNRs) could generate a series of distinct colors that covers nearly the whole visible range from 400 to 760 nm. These vivid colors can be easily distinguished by the naked eye; as a result, the accuracy of visual inspection could be greatly improved. Next, we demonstrate the utility of AuNRs as multicolor chromogenic substrate to develop a number of colorimetric immunoassay methods, e.g., multicolor enzyme-linked immunosorbent assay (ELISA), multicolor competitive ELISA, and multicolor magnetic immunoassay (MIA). These methods allow us to visually quantify the concentration of a broad range of target molecules with the naked eye, and the obtained results are highly consistent with those state-of-the-art techniques that are tested by the sophisticated apparatus. These multicolor portable and cost-effective immunoassay approaches could be potentially useful for a number of applications, for example, in-home personal healthcare, on-site environmental monitoring, and food inspection in the field.