Cubic NaYF4 single crystals co-doped with ~1.90 mol% Ho3+ and various Tm3+ concentrations (0.99mol%, 1.58 mol%, 2.37 mol%, 3.16 mol%, 3.99 mol%, 7.19 mol%) were grown by using the Bridgman method. The energy transfer from Tm3+ to Ho3+, concentration quenching of Tm3+ and the optimum fluorescence emission around 2.04 μm of Ho3+ ion were investigated based on the measured absorption spectra, emission spectra, emission cross section and decay curves under excitation of 800 nm LD. The emission intensity at 2.04 μm increased with the increasing of Tm3+ concentration from 0.99 mol% to 1.59 mol%, and decreased with the increasing of Tm3+ concentration from 1.59 mol% to 7.19 mol% when the concentration of Ho3+ was held constantly at ~1.90 mol%. Moreover, the maximum emission cross section reached to 2.17×10－20 cm2 and the maximum fluorescence lifetime was 21.72 ms for Ho3+(1.90 mol%)/Tm3+(1.59 mol%) co-doped one. At the same time, the energy transfer coefficients of this sample were calculated based on the absorption cross-section of Ho3+ and the emission cross-section of Tm3+, which is CTm-Ho=21.14×10－40 cm6/s and CHo-Tm=2.05×10－40 cm6/s respectively.