Effect of interrupted cooling time on microstructure transformation, precipitation behaviour and hardness of a Ti-V-Mo microalloyed steel was investigated by means of OM, SEM, TEM and Vickers hardness tester, and the factors influencing the hardness change were discussed. The results reveal that when the Ti-V-Mo microalloyed steel after austenitization is isothermally cooled at 630°C from 0 to 3 h, with the increase of interrupted time, the proportion of ferrite in the matrix increases while the proportion of martensite and bainite decreases. In addition, the hardness increases first and then becomes stable, then re-increases and finally declines slightly. At the interrupted time ranging from 60 to 1200 s, the hardness plateau occurs because the precipitation strengthening effect of (Ti, V, Mo) C nano-particles can compensate for the hardness loss caused by matrix softening due to phase transformation. When the interrupted time is 3600 s, the maximum hardness is 457HV, and the precipitation strengthening effect of (Ti, V, Mo) C nanoparticles is the best.