Semiconducting polymer nanoparticles (Pdots) have been demonstrated to be a promising class of probes for use in fluorometric immunochromatographic test strips (ICTS). The advantages of Pdots in ICTSs include ultrahigh brightness, minimal nonspecific adsorption, and multicolor availability, which together contribute to the high sensitivity, good specificity, and multiplexing ability. These unique properties can therefore circumvent several significant challenges of commercial ICTSs, including insufficient specificity/sensitivity and difficulty in quantitative and multiplexed detection. Here, we developed a colorimetric and fluorescent bimodal readout ICTS based on gold-Pdot nanohybrids for the determination of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA 21-1) expressed abnormally in human blood of non-small-cell lung cancer (NSCLS). The vivid color from Au nanomaterials can be used for rapid qualitative screening (colorimetry) in 15 min, while the bright fluorescence of Pdots is ideal for the advanced quantitative measurements of CEA and CYFRA21-1 concentrations in whole blood samples. This bimodal ICTS platform possesses phenomenal detection sensitivity of 0.07 and 0.12 ng/mL for CYFRA21-1 and CEA, respectively. The accuracy and reliability of this ICTS platform were further evaluated with clinical serum samples from NSCLS patients at different stages, showing good consistency with the results from electrochemiluminescence immunoassay.