In vivo multiplexed bioimaging in the second near-infrared window (NIR-II, 1000–1700 nm), owing to its superiorities of simultaneous multi-channel (multicolor) observations for multiple intrinsic biomarkers, high sensitivity, deeper penetration and high spatiotemporal resolution, and high throughput, has been a growing technology in fundamental medical diagnosis and clinical applications. Among several NIR-II nanoprobes for multiplexed bioimaging, the inorganic nanoprobes based on quantum dots (QDs) and lanthanide downconversion nanoparticles (DCNPs), as well as organic fluorophores based on donor−acceptor−donor (D−A−D) chromophores, polymethine dyes, and lanthanide complexes are extensively suitable for intravital imaging and diagnosis of disease, exhibiting excellent accomplishments. Here, we summarize recent advances in NIR-II-emitted nanoprobes for intravital multiplexed bioimaging. Furthermore, the current challenges and potential opportunities in designing novel long-wavelength nanoprobes for deep-tissue intravital multiplexed bioimaging are discussed.