Ultrasensitive fluorescence imaging of proteins on western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CN-PPV) is demonstrated. A detection limit at the single-picogram level in dot blots is achieved; with conventional western blotting, 50 pg of transferrin and trypsin inhibitor after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer onto a polyvinylidene fluoride membrane are detected. This method does not require any additional equipment or time compared with the conventional procedure with traditional fluorescent probes.