Bilirubin is an important biomarker of jaundice, and the quantitative detection of bilirubin is of great significance in the diagnosis and prevention of early jaundice. In this paper, Fe3+ quenched conjugated polymer nanoparticles were used as a "turn-off-on" fluorescence platform to detect bilirubin for the first time. The CPNsPBMC-COOH were prepared through a nanoprecipitation method based on polystyrene maleic anhydride and PBMC, a conjugated polymer synthesized by our research group. The fluorescence of CPNsPBMC-COOH can be efficiently quenched closely to 95% by Fe3+. The optimized fluorescence platform was obtained (CPNsPBMC-COOH: 10 μg/mL, Fe3+: 4.5 μM). The platform to detect bilirubin has a higher sensitivity with a 5 nM of LOD. The detection range was in the 1.32–26.68 μM, which can cover the concentration of bilirubin in normal adult serum (<25 μM). Moreover, the stoichiometries and binding constants of CPNsPBMC-COOH, bilirubin, and Fe3+ are obtained. The binding ability is better for bilirubin and Fe3+. The static quenching mode is found by the absorption spectra and the fluorescence lifetimes. Lastly, this method has obtained satisfactory results in real biological samples (mouse serum and mouse plasma). This work can be used for daily rapid clinical bilirubin detection.