High-performance 24CrNiMo steel was fabricated using Laser Powder Bed Fusion (LPBF). Subsequent quenching treatment was applied and the influence of quenching temperatures on micro-structure evolution and properties was systematically characterised and analysed. The micro-structure of the as-built steel consisted of two parts. The first part comprised martensite with twins combined with ω-Fe nano-particles, and the second part consisted of lower bainite in the molten pool, as well as upper bainite, granular bainite and tempered martensite in the heat-affected zone. With the quenching temperatures varying from 800 °C to 950 °C, the micro-structure gradually transformed from acicular ferrite + martensite to tempered martensite +θ-Fe3C carbides, and the grain size exhibited noticeable growth. Moreover, quenching treatments could eliminate the anisotropy and inhomogeneity of the micro-structure. The rod-shaped nanosized η-Fe2C and θ-Fe3C precipitates were clearly observed, which were converted from ω-Fe and distributed at multiple angles in the lath. The size and number of nano-precipitates, triggered by the high self-tempering degree of martensite, gradually increased. The relationships among grain size, the twins, dislocation density and nano-precipitation and the dramatically improved performance of quenched samples were analysed using strengthening mechanisms. After quenching at 850 °C, the as-built 24CrNiMo steel attained ultra-high mechanical properties including hardness, Ultimate Tensile Strength (UTS), Elongation (El) and impact energy with values of 480.9 HV1, 1611.4 MPa, 9.8% and 42.8 J, respectively. Meanwhile, both the wear and thermal fatigue resistance increased by approximately 40%. This study demonstrated that LPBF-fabricated 24CrNiMo steel, with matching good performances, can be achieved using a subsequent one-step quenching process.