The invention provides a preparation method of a fluorescent probe for identification of microfilament bacteria. The method first uses halogenated long carbon chain compound to modify 4-methyl pyridine part of a carbazole pyridine styrene cyanine dye, and then the dye reacts with 3-formyl-N-ethyl carbazole for the synthesis of the fluorescent probe with a long hydrophobic chain. The invention has the beneficial effect that modification and preparation of the carbazole pyridine styrene cyanine dye are synchronized in the probe preparation process, the preparation method is simple, and the prepared fluorescent probe has little background interference, high fluorescence intensity and stable fluorescent property. The characteristic of hydrophobic surface of microfilament bacteria is utilized that the prepared fluorescent probe with long hydrophobic chain in this application can combined with microfilament bacteria to achieve the purpose of fluorescent identification of microfilament bacteria.