A new cationic poly(phenylene vinylene) derivative (PPV-NMe3+) is synthesized, and it exhibits a differential binding ability to microbial cell walls with different components. By varying the ion strengths of the buffer solution, single PPV-NMe3+ molecules can discriminate fungi, Gram-positive bacteria, and Gram-negative bacteria in a rapid and simple manner. Thus, cationic conjugated polymers exhibit high potential as a diagnostic material for the detection and discrimination of pathogens.