Fluorescence imaging in the second near-infrared window (NIR-II, 1000−1700 nm) has emerged as a promising choice for in vivo imaging and has attracted considerable attention in the past decade. In contrast to conventional optical imaging in the visible region and NIR-I (750−1000 nm) window, NIR-II fluorescence imaging exhibits reduced photon scattering, absorption, and autofluorescence from biological tissues, thereby enabling deeptissue penetration and high-resolution bioimaging. Among the various kinds of NIR-II optical materials, quantum dots (QDs) have flourished the field, propelling it into a development stage. Their superior traits include tunable emission spanning the entire NIR-II window and abundant surface functionalities that facilitate the ingenious design of specific and highly sensitive QDs nanoprobes, further expanding their biological application potential. In this review, we first introduce the different types of NIR-II QDs and highlight the design strategies of functional QDs-based nanoprobes. Then, the recent advances using QDs nanoprobes for NIR-II bioimaging, biosensing, and imaging-guided therapy applications are summarized in detail. Finally, the broad prospects and significant challenges to advance NIR-II QDs for clinical disease diagnosis and treatment are discussed.