A novel Na5Y9F32 single crystals tri-doped with fixed ~0.8 mol% Ho3+, ~1 mol% Yb3+ and various Pr3+ from 0 to 0.8 mol% were successfully grown by a modified Bridgman method for 2.9 μm mid-infrared application. The effects of Pr3+ ion on the 2.9 μm emission was investigated systematically with the help of the absorption spectra, emission spectra and fluorescence decay curves. The emission intensity at ~2.9 μm of Ho3+: 5I6 → 5I7 was enhanced gradually as increase of Pr3+ concentration from 0 to 0.8 mol% under excitation of 980 nm laser diode (LD). The maximum emission cross-section at ~2.9 μm was calculated to be 5.62 × 10−20 cm2 according to the measured emission spectra. The energy transfer mechanism among Ho3+, Yb3+ and Pr3+ was studied. When the Pr3+ ion concentration reached 0.8 mol%, the energy conversion efficiencies for Ho3+: 5I7 → Pr3+: 3F2 and Ho3+: 5I6 → Pr3+: 3F4 were estimated about ~51.76% and ~15.73%, respectively. A remarkably augmented conversion process of Ho3+: 5I7 → Pr3+: 3F2 by the introduction of Pr3+ ion into Ho3+/Yb3+ system results into the enhancement of ~2.9 μm emission. The Pr3+ ion takes as an effective deactivation of the Ho3+ ion in the Na5Y9F32 single crystal. The Pr3+/Ho3+/Yb3+ tri-doped Na5Y9F32 single crystals may become a new choice for ~2.9 μm mid-infrared solid-state laser due to its both excellent optical properties and chemical stability.