The metastable α' phase and dislocation characteristics (e.g., density and constituents) are of vital importance for the mechanical responses of (α+β) titanium alloys. In this work, to reveal the in-depth decomposition mechanisms of dislocations and metastable α' and their influences on mechanical properties in a Ti-6Al-4V (α+β) alloy, the thermal stability of different microstructures tailored by various cooling approaches were investigated utilizing scanning electron microscope, electron backscattered diffraction, transmission electron microscope and X-ray diffraction line profile analysis. The results showed that the initial characteristics of α' and α laths and dislocation density were influenced by the cooling methods remarkably. The thermal stability of Ti-6Al-4V alloy increased with decreasing cooling rate. The improvement in thermal stability can be ascribed by the decrement in dislocations, partitioning of the alloying elements and grain orientations variation of the α' lath and α phase. It is also found that the plastic strain accommodation of β→α' transformation was dominated by dislocations. During stabilization annealing treatments, the , and dislocations simultaneously decomposed. The decomposition of dislocations and metastable α' phase during various stabilization annealing and the particular twins and stacking faults microstructures formed during quenching have a great influence on the properties of the studied Ti-6Al-4V alloy.