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There have been enormous efforts for developing the next generations of fluorometric lateral flow immunochromatographic strip (ICTS) owing to the great advances in fluorescent materials in these years. Here we developed one type of fluorometric ICTS based on ultrabright semiconducting polymer dots (Pdots) in which the traffic light-like signals were created by energy transfer depending on the target concentration. This platform was successfully applied for qualitatively rapid screening and quantitatively precise analysis of prostate-specific antigen (PSA) in 10 min from merely one drop of the whole blood sample. This FRET-created traffic light ICTS possesses excellent specificity and an outstanding detection sensitivity of 0.32 ng/mL for PSA. Moreover, we conducted proof-of-concept experiments to demonstrate its potential for multiplexed detection of cancer biomarkers at the same time in an individual test strip by taking advantage of the traffic light signals. To the best of our knowledge, it is the first model of a traffic light-like immunoassay test strip based on Pdots with multiplexing ability. These results would pave an avenue for designing the next generation of point-of-care diagnostics.