As fluorescence in the second near-infrared window (NIR-II, 1000-1400 nm) could image deep tissue with high signal-to-noise ratios compared with that in NIR-I (750-900 nm), Ag2Se quantum dots (QDs) with fluorescence in the NIR-II could be ideal fluorophores. Here, we described a biosynthesis method to prepare the Ag2Se QDs by using temporally coupling the irrelated biochemical reactions, whose photoluminescence (PL) emission can reach NIR-II. The nanoparticles were characterized by transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results showed that the nanoparticles obtained by extracellular purification were Ag2Se QDs with a uniform size of 3.9 +/- 0.6 nm. In addition, the fluorescence intensity of Saccharomyces cerevisiae was improved successfully by nearly 4-fold by constructed engineering strain. In particular, the biosynthesis of Ag2Se QDs had good biocompatibility because it was capped by protein. Furthermore, investigating the toxicity of Ag2Se on cells and NIR images of nude mice showed that the Ag2Se synthesized using S. cerevisiae had low toxicity and could be used for in vivo imaging. In this work, the synthesis pathway of biocompatible Ag2Se was broadened and laid a foundation for the enlarged applicability of bioimaging in the biosynthesis of NIR-II QDs. (C) 2021 Elsevier Inc. All rights reserved.