An enzyme-linked immunosorbent assay that is dependent on enzyme amplification has dominated the current field of protein detection; however, limited multiple detection ability and susceptible enzymatic reactions, and low sensitivity may severely hinder its application. Here, we report a new signal amplification scheme based on allochroic molecule modified carboxyl graphene oxide (cGO), which can be used to develop a multicolor immunoassay named as allochroic-cGO linked immunosorbent assay (ALISA). Thanks to high adsorption levels and a wide selection of allochroic molecules, the simultaneous colorimetric detection of diagnostic biomarkers at a picogram level can be successfully achieved for the first time. In addition, the color change triggered by acidic or basic water can provide a simple, rapid, stable and economical signal output, further meeting the growing biodetection requirements. Moreover, with the help of ALISA, we demonstrate that the combined detection of three tumor biomarkers, including carcino-embryonic antigen, neuron-specific enolase, and cytokeratin-19 fragment, is more valuable for differentiating lung cancer patients than the detection of a single biomarker, further manifesting the superiority of ALISA. All in all, this straightforward approach not only opens up new prospects for multicolor immunoassays, but also has great potential for applications in resource-constrained settings.