Abstract: In order to develop a quick method for detecting the Escherichia coli (E. coli) in cigarette paper, pH-sensitive fluorescent dye fluorescein isothiocyanate (FITC) was coupled with amino modified DSPE-PEG2000 to form fluorescent self-assembled nano-micelles in phosphate buffer saline (PBS) and used to prepare pH-sensitive fluorescent sensory gel. The fluorescent sensors were thus developed by separating fluorescent sensory gel from liquid culture medium for E. coli using aqueous phase filter membrane, and the quick detection of E. coli was realized via monitoring the change of physiological pH values during the growing procedure of E. coli. The results showed that:1) The sizes of nano-micelles ranged from 100 to 500 nm, excitation and emission wavelengths were 480 and 520 nm respectively. The fluorescent intensity of the sensory gel heightened when pH values raised from 4.5 to 9.0. 2) On the basis of traditional culture theory, fluorescent sensors could be used for the quick detection of E. coli in cigarette paper within much shorter screening time compared with traditional plate counting method. This method is suitable for assessing the amount of E. coli in cigarette paper.