Upconversion nanoparticles (UCNPs) with multi-color emissions have been studied extensively owing to their potential applications in diverse research fields. The upconversion luminescence (UCL) of UCNPs is greatly affected by the excitation mode or intensity of NIR laser. Herein, the effect of excitation mode (continuous wave and pulsed excitation) on the UCL and modulation mechanism of β-NaYF4:Yb/Er nanocrystals were investigated. In this Yb3+-Er3+ co-doped nanocrystals, temporally red emission was dramatically realized by employing pulsed excitation mode. As a result, bright red color luminescence was output in β-NaYbF4:2%Er3+ nanocrystals. Besides, pulsed excitation can partially eliminate the concentration quenching effect, enhancing the optimal Yb3+ doping ratio from 20% to 50%. Excitation mode would change the dynamic balance of excitation energy transfer processes, which eventually leads to the variation of UCL of β-NaYF4:Yb/Er. These fantastic insights may open up new pathways to efficiently tailor upconversion luminescence performance in favor of further development of applications.