The metastable ω and α″ in titanium alloys have a significant impact on the subsequent α precipitates and the final mechanical properties. Herein, the microstructure transformation of coexisting ω/α″ during aging and their effects on tensile properties have been systematically investigated in Ti-3Al-5Mo-4.5 V (wt%) α/β titanium alloy. The results demonstrate that the secondary phase within the β-transformed (βtrans) grain evolves from embryonic ω + α″ to isothermal ω + α″, and ultimately transforms to secondary α (αs) with the aging temperature ascending from 300 °C to 550 °C. As embryonic ω transforms to isothermal ω, the yield strength increases from 1130.0 ± 14 MPa to 1259.5 ± 5.5 MPa with weak elongation due to the excellent strengthen effect of undeformed isothermal ω. After aging at 550 °C, coexisting micron-scale αp and nanoscale αs microstructures lead to enhanced elongation (increase by 93.7 %), sacrificing only 21.7 % yield strength. Both <a> and <c + a> dislocations are simultaneously presented in micron-scale αp grain, while nanoscale αs plates act as effective obstacles for dislocation motion resulting in accumulation of dense dislocations near the β/αs interfaces. This work provides new insights on strengthening strategy and developing new Ti-alloys.