ABSTRACT A novel dicationic Quinoline-Carbazole fluorescent probe with hydrophobic long-chain alkane was designed and synthesized based on the property of Microthrix parvicella (M. parvicella) in situ utilizing long-chain fatty acids (LCFA) in the activated sludge system. 1H NMR spectrum, ultraviolet–visible (UV-Vis) absorption spectra, and fluorescent spectra analysis demonstrated that the probe was successfully obtained. The probe had a large stokes-shift ranging from 102 to 144 nm in different solvents, which were benefit for the fluorescent labelling properties. The labelling experiment indicated that the prepared probe could absorb onto the surface of M. parvicella through hydrophobic bond. Much stronger yellow fluorescence of M. parvicella was observed at the concentration of 1.0 × 10−5 mol/L when compared with the zooglea, which makes it easy to distinguish M. parvicella from the zooglea. In addition, the photostability of the probe was also investigated, and the result showed that the probe was quite stable in a long period of time. All the results indicated that the prepared probe was suitable for the labelling of M. parvicella.