Cationic poly(fluorene-co-phenylene) derivative (PFP-NMe3+) forms a supramolecular complex with cucurbituril (CB), which could be reversibly disassembled by amantadine (AD) to release PFP-NMe3+ due to the formation of more stable CB/AD complex. The cationic PFP-NMe3+ is an amphiphilic structure and could bind to negatively charged membrane of pathogen by multivalent interactions. Upon the formation of PFP-NMe3+/CB complex, the CB could bury the side-chain alkyl groups and decreases the hydrophobic interactions of PFP-NMe3+ on the surface of pathogens; thus, PFP-NMe3+ exhibits different interaction modes with pathogens before and after assembly with CB. The PFP-NMe3+/CB supramolecular complex could be explored as optical sensor for simple, rapid, and in situ detection and discrimination of multiple pathogens by taking advantage of optical signal changes of PFP-NMe3+/CB complex before and after disassembly by AD on the pathogen surfaces. The new sensor can realize in situ detection and identification of Gram-negative bacteria (E. coli, P. aeruginosa), Gram-positive bacteria (B. subtilis, S. aureus, E. faecalis), and the fungi (C. albicans, S. cerecisiae) and can also discriminate different strains of the same species. Blend samples of these pathogens could be identified successfully as well. In comparison with conventional blood culture-based pathogen assay methods that require at least for 24 h, the PFP-NMe3+/CB complex only needs 2 h (including pathogen culture, pathogen harvest by centrifuging, and optical assay procedures) to stratify diverse pathogen types and also does not require specific biomarkers or cell labeling.