The effects of different tempering temperatures on the microstructure evolution and mechanical properties of the new low-alloy ultra-high-strength 45CrNiSiMnMoVA steel after quenching were investigated by mechanical property tests, SEM and TEM. The results show that a complex phase organization consisting of martensite/ lower bainite of the tested steel after treated at 920°C×1h+(320~380)°C×4h was obtained, and the partition interface of the lath martensite bundle became blurred from clear with the increase of tempering temperature; In the proposed tempering temperature range, the toughness of the alloy has become better while maintain the strength without decreasing basically, and when the tempering temperature is 350°C, the alloy has the optimal comprehensive mechanical properties of strength, plasticity and toughness together. The analysis concluded that the strong toughening of the tested steel was mainly attributed to the coupling effect of the alloying elements in the steel and the composite toughening of the nano-precipitated phases, among other aspects.