Fluorescence imaging with high spatiotemporal resolution and sensitivity is employed for in vivo visualization and detection applications. Compared with visible light and the first near-infrared window (700–900 nm), the second near-infrared window (1 000–1 700 nm) imaging has a lower auto-background fluorescence, deeper tissue penetration, and a higher signal-to-noise ratio, thus highlighting its broad prospects in the biomedical field. Currently, reported second near-infrared region probes include single-walled carbon nanotubes, rare-earth nanoparticles, quantum dots, metal materials, and organic molecular dyes. Multimodal imaging can overcome the limitations of single imaging and provide more comprehensive information on the anatomical structure, tissue composition, and cellular and molecular quantification of lesions to achieve multi-level visualization. Therefore, second near-infrared window nanoprobes must be engineered for multimodal imaging. Moreover, these nanoprobes can be actively targeted by modification with antibodies, peptides, nucleotides, or biofilms to obtain detailed and accurate information on biological systems. This review describes the active targeting capabilities of various second near-infrared window nanoprobes in multimodal imaging, diagnosis, and treatment of different diseases by carrying different ligands and the common features and future application prospects of second near-infrared window fluorescent nanoprobes with targeting ability.