Accurate intraoperative tissue identification is critical to tumor surgery. However, conventional methods are labor‐ and time‐intensive, which greatly delay the intraoperative decision‐making. Herein, a matrix metalloproteinase (MMP)14‐activated NIR‐II nanoprobe (A&MMP@Ag2S‐AF7P) is presented for rapid unperturbed‐tissue analysis for ex vivo and in vivo neuroblastoma diagnosis. A&MMP@Ag2S‐AF7P displays negligible fluorescence in normal tissues but is activated quickly by inhibiting the fluorescence resonance energy transfer (FRET) between Ag2S QDs and A1094 mediated by MMP14 overexpressed in neuroblastoma; meanwhile, the exposure of the membrane penetrating peptide R9 (TAT‐peptide) results in efficient internalization of nanoprobes in the cancer cells, providing superior tumor‐to‐normal (T/N) tissue ratio. Instant illumination of the lesion and well‐defined tumor margins make the nanoprobes a suitable rapid diagnostic reagent for cancer surgical or tissue biopsy procedures. [ABSTRACT FROM AUTHOR]