The maintenance of an intact membrane structure is of great importance for bacteria to execute various biological functions. However, chemical probes for monitoring the dynamic changes of bacterial membranes are barely reported. Herein, we, for the first time, report a novel polarity-sensitive probe for reflecting the packing degree of bacterial membrane lipids. Specifically, we synthesize a membrane-targeting fluorescent probe (TICT-lipid) that possesses both twist intramolecular charge transfer and aggregation-induced emission properties. TICT-lipid exhibits sensitive responses to the minute difference in the packing degree of membrane lipids, facilitating rapid differentiation of Gram-negative and Gram-positive bacteria. Interestingly, in the presence of membrane-disrupting antibiotics, the localization of TICT-lipid shifts from the outer membrane to the cell membrane by outputting blue-shifted and enhanced emission, making the mechanism of action of antibiotics clearly visible. TICT-lipid is a polarity-sensitive fluorescent probe, holding great promise in the study of membrane-related bacterial processes and antibiotic screening.