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Abstract Rapid detection and quantification of outer membrane vesicle (OMV) are of both scientific value and clinical implications. However, limited tools are available for investigations of OMVs. Herein, we report a novel fluorescent probe with aggregation‐induced emission (AIE) characteristics, namely, OEO‐TPE‐MEM (OTM), for OMV detection. OTM emits faintly in an aqueous medium, but its fluorescence could be effectively turned on upon interacting with bacteria bodies and OMVs produced by Gram‐negative bacteria. Notably, OTM could provide quantitative information on bacterial membrane remodeling and OMV secretion and be applied to high‐throughput screening of OMV‐inducing agents. This study presents a powerful AIE probe for imaging and quantitative analysis of bacteria envelop and derived OMVs, which might be applied for evaluating research and clinical antimicrobial materials in future studies.