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Er3+-sensitized upconversion nanoparticles (UCNPs) have attracted great attention due to their tunable upconversion (UC) emissions, low cytotoxicity, high resistance to photobleaching and especially multiple effective excitation wavelengths. However, detailed energy conversion between Er3+ and Tm3+ ions in Y2O3 UCNPs is still a problem, especially under multi-wavelength and variable pulse width excitation. In this work, we successfully fabricated a series of Er3+-sensitized Y2O3 nanocrystals by a spray flame synthesis method with a production rate of 40.5 g h&minus, 1. The as-prepared UCNPs are a pure cubic phase with a mean size of 14 nm. Excited by both 980 and 808 nm lasers, the tunable upconversion luminescence (UCL) from Er3+ ions was achieved by increasing the Er3+ doping concentration, co-doping Tm3+ ions and extending excitation pulse-width. The investigations of the lifetimes and the laser power dependence of UC emissions further support the proposed mechanism, which provides guidance for achieving effective color control in anticounterfeiting and multiplexed labeling applications. In addition, the red UC emission at about 5 mm beneath the tissue surface was observed in an ex vivo imaging experiment under the excitation of 808 nm laser, indicating that the Y2O3:Er3+/Tm3+ UCNPs have great prospects in further biological applications.