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The development of efficient fluorescence-based detection tools with high contrast and accuracy in cancer diagnosis has recently attracted extensive attention. Changes in the microenvironments between cancer and normal cells provide new biomarkers for precise and comprehensive cancer diagnosis. Herein, a dual-organelle-targeted probe with multiple-parameter response is developed to realize cancer detection. We designed a tetraphenylethylene (TPE)-based fluorescent probe TPE-PH-KD connected with quinolinium group for simultaneous detection of viscosity and pH. Due to the restriction on the double bond's rotation, the probe respond to viscosity changes in the green channel with extreme sensitivity. Interestingly, the probe exhibited strong emission of red channel in acidic environment, and the rearrangement of ortho-OH group occurred in the basic form with weak fluorescence when pH increased. Additionally, cell colocalization studies revealed that the probe was located in the mitochondria and lysosome of cancer cells. Following treatment with carbonyl cyanide m -chloro phenylhydrazone (CCCP), chloroquine, and nystatin, the pH or viscosity changes in the dual channels are also monitored in real-time. Furthermore, the probe TPE-PH-KD could effectively discriminate cancer from normal cells and organs with high-contrast fluorescence imaging, which sparked more research on an efficient tool for highly selectively visualizing tumors at the organ level. [Display omitted] • A novel TPE-based fluorescent probe for detection of pH/viscosity in dual channels was developed. • Probe could monitor pH/viscosity fluctuation upon stimulation with CCCP, chloroquine and nystatin. • Probe could distinguish tumor and normal cells and tissues in high contrast by fluorescent imaging. [ABSTRACT FROM AUTHOR]