A single-frequency pulsed holmium-doped yttrium lithium fluoride (Ho:YLF) amplifier pumped by a Tm-doped fiber laser was demonstrated. The seed was an injection-seeded Q-switched Ho:YLF laser. The output energy from the single-frequency pulsed amplifier was 24.2 mJ, with a pulse width of 250 ns at a pulse repetition frequency (PRF) of 100 Hz. The energy stability during 30 min was improved to 1% after the single-frequency pulsed Ho:YLF laser was amplified. The line width of the single-frequency pulsed spectrum of the Ho:YLF amplifier was 2.81 MHz. The single-frequency pulsed Ho:YLF amplifier can be applied to differential absorption lidar (DIAL), since its output spectrum is around the P12 CO₂ absorption line.