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.