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Polyols are important biomass-based building blocks for chemical production. In this work, a hydrotalcite-derived PtNi alloy catalyst (PtNi/HT) was prepared with high metal dispersion under microwave-assisted synthesis and employed for glucose hydrogenation, where 95.7% of glucose was converted with 90.1% selectivity into polyols at 393 K in water. The PtNi/HT was stable and could be recycled for at least five runs for glucose. Lower reaction temperatures (333–373 K) contributed to glucose hydrogenation to hexitols, while higher reaction temperatures (373–413 K) facilitated the C–C activation of glucose to generate more polyols with less carbon (C2–C4). The Ptδ−–Niδ+ pair on PtNi/HT contributed to both H2 activation and adsorption of the aldehyde group in glucose hydrogenation. The basic support of PtNi/HT can efficiently catalyze the glucose isomerization and retro-aldol reaction in the aqueous phase. This study advocates a green approach for sustainable biorefinery of readily available glucose into value-added chemicals.