Fluorescence imaging with high sensitivity and minimal invasiveness has received tremendous attention, which can accomplish visualized monitoring and evaluation of cancer progression. Compared with the conventional first near‐infrared (NIR‐I) optical window (650–950 nm), fluorescence imaging in the second NIR optical window (NIR‐II, 950–1700 nm) exhibits deeper tissue penetration capability and higher temporal‐spatial resolution with lower background interference for achieving deep‐tissue in vivo imaging and real‐time monitoring of cancer development. Encouraged by the significant preponderances, a variety of multifunctional NIR‐II fluorophores have been designed and fabricated for sensitively imaging biomarkers in vivo and visualizing the treatment procedure of cancers. In this review, the differences between NIR‐I and NIR‐II fluorescence imaging are briefly introduced, especially the advantages of NIR‐II fluorescence imaging for the real‐time visualization of tumors in vivo and cancer diagnosis. An important focus is to summarize the NIR‐II fluorescence imaging for deep‐tissue biomarker analysis in vivo and tumor tissue visualization, and a brief introduction of NIR‐II fluorescence imaging‐guided cancer therapy is also presented. Finally, the significant challenges and reasonable prospects of NIR‐II fluorescence imaging for cancer diagnosis in clinical applications are outlined.

DNAzyme shows great promise in designing a highly sensitive and specific sensing platform; however, the low cellular uptake efficiency, instability, and especially the insufficient cofactor supply inhibit the intracellular molecule sensor applications. Herein, we demonstrate a novel type of DNAzyme-based self-driven intracellular sensor for microRNA (miRNA) detection in living cells. The sensor consists of a metal–organic framework [zeolite imidazole framework (ZIF-8)] core loaded with a shell consisting of a rationally designed DNAzyme, where the substrate strand is modified with FAM and BHQ-1 nearby both the sides of the restriction site, respectively, while the enzyme strand consists of two separate strands with a complementary fragment to the substrate strand and the targeting miRNA, respectively. The ZIF-8 nanoparticles enable the efficient delivery of DNAzyme into the cell and protect the DNAzyme from degradation. The pH-responsive ZIF-8 degradation is accompanied with the release of the DNAzyme and Zn2+ cofactors, and the intracellular target miRNAs recognize and activate the DNAzyme driven by the Zn2+ cofactors to cleave the substrate strand, resulting in the release of the FAM-labeled shorter product strand and increased fluorescence for miRNA detection. The self-driven approach can be generally applied to various miRNAs’ detection through DNAzyme engineering.