Owing to their low density, high specific strength, biocompatibility, and good corrosion resistance, titanium and its alloys have been widely used in the aerospace, biomedical, and marine engineering fields. As engineering applications of titanium alloys continue to develop, especially in special engineering projects, the service safety and stability of titanium alloys must be satisfied under extremely complex conditions. Unfortunately, traditional titanium alloys usually exhibit low plastic-deformation ability and no significant work hardening behavior, which limits their applicability. Improving both the strength and ductility of these materials is expected to broaden their engineering applications. In recent years, β-type (body-centered cubic) titanium alloys have shown good formability and impact toughness, by virtue of their diverse plastic deformation modes and excellent ageing strengthening. Therefore, they are promising candidates for titanium alloys with a good strength-ductility combination. In this work, a multilayered Ti-10Mo-1Fe/3Fe alloy was manufactured by a multi-pass hot rolling and heat treatment, and the coupling effects of pre-strain and isothermal ageing on the mechanical properties of the alloy were studied by various techniques: laser scanning confocal microscopy, XRD, SEM, SEM-EDS, EBSD, a Vickers hardness tester, and a tensile testing machine. After pre-strain and isothermal ageing, the alloy exhibited {332}<113> twins and slip bands alternately multilayered deformation microstructures. The alloy demonstrated a relatively high yield strength and large uniform elongation. The high yield strength resulted from the initial plastic deformation, which was dominated by dislocation slips due to isothermal ω-phase precipitation. The early onset of plastic instability after yielding was hindered by the pre-strain induced twins, and the uniform elongation was enhanced not only by the dynamic Hall-Petch effect caused by further twinning activation, but also by interactions between the twin and layer-interface. As demonstrated on this multilayered alloy with twinning and dislocation-slip coupled deformation, the strength-ductility combination in β-type titanium alloys can be controlled through the coupling effect of pre-strain induced {332}<113> twins and the subsequently precipitated ω phase.