Quantum dots (QDs) with fluorescence in the second near-infrared window (NIR-II, 1000-1400 nm) are ideal fluorophores for in vivo imaging of deep tissue with high signal-to-noise ratios. Ag₂Se (bulk band gap 0.15 eV) is a promising candidate for preparing NIR-II QDs. By using 1-octanethiol as ligand to effectively balance the nucleation and growth, tuning the fluorescence of Ag₂Se QDs was successfully realized in the NIR-II window ranged from 1080 to 1330 nm. The prepared Ag₂Se QDs can be conveniently transferred to the aqueous phase by ligand exchange, showing great potential for multicolor NIR-II fluorescence imaging in vivo.