Biosynthesis of metal nanoparticles represents a clean, eco-friendly and sustainable green chemistry engineering. Lately, a number of metal selenides were successfully synthesized by biological methods. Here, cuprous selenide (Cu2Se) nanospheres were prepared under mild conditions by a novel biological-chemical coupling reduction process. The simple process takes place between EDTA-Cu and Na2SeO3 in presence of an alkaline solution containing NaBH4 and a selenite-reducing bacteria, Pantoea agglomerans. It is noteworthy that the isolated Pantoea agglomerans and Cu+ ions, where the latter are obtained from reducing Cu2+ ions by NaBH4, play a key role, and Cu+ ions not only can promote the generation of Se2- ions as a catalyst, but also can react with Se2- ions to form Cu2Se. XRD pattern, SEM, and TEM images indicated that Cu2Se nanoparticles were tetragonal crystal structure and the nanospheres diameter were about 100 nm. EDX, UV-vis, and FTIR spectra show that the biosynthesized Cu2Se nanospheres are wrapped by protein and have a better stability. This work first proposes a new biosynthesis mechanism, and has important reference value for biological preparation of metal selenide nanomaterials. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1264-1270, 2016