Na5Y9F32 single crystals doped with ～0.8-mol％ Ho3+, ～1-mol％ Tm3+, and various Er3+ ion concentrations were prepared by a modified Bridgman method. The effects of Er3+ion concentration on 2.0-μm emission excited by an 800-nm laser diode were investigated with the help of their spectroscopic properties. The intensity of 2.0-μm emission reached to maximum when the Er3+ ion concentration was～1 mol％. The energy transfer mechanisms between Er3+, Ho3+, and Tm3+ ions were identified from the change of the absorption spectra, the emission spectra, and the measured decay curves. The maximum 2.0-μm emission cross section of the Er3+/Ho3+/Tm3+ tri-doped Na5Y9F32 single crystal reached 5.26 × 10?21 cm2. The gain cross section spectra were calculated according to the absorption and emission cross section spectra. The cross section for～2.0-μm emission became a positive gain once the inversion level of population was reached 30％. The energy transfer efficiency was further increased by 11.81％through the incorporation of Er3+ion into Ho3+/Tm3+ system estimated from the measured lifetimes of Ho3+/Tm3+-and Er3+/Ho3+/Tm3+-doped Na5Y9F32 single crystals. The present results illustrated that the Er3+/Ho3+/Tm3+tri-doped Na5Y9F32 single crystals can be used as promising candidate for 2.0-μm laser.