Face-centered cubic (FCC) medium entropy alloys (MEAs) have received considerable attention due to their impressive mechanical properties and responses. However, their practical application is limited by their modest yield strengths. The potential enhancement of the mechanical properties of single-phase MEAs was explored in this study through a synergistic approach combining heterogeneous structure design with subsequent cryo-pre-straining. A heterogeneous lamella structure was produced in a single-phase Fe55Mn20Cr15Ni10 MEA via two-step rolling and annealing. Cryo-pre-straining at varying degrees (6, 12, 21, and 36%) introduced hexagonal close-packed (HCP) phase, high-density dislocations, twins, and stacking faults, leveraging the reduced stacking fault energy at cryogenic temperatures. This process enhanced the alloy's yield strength from 353 MPa to 1.2 GPa (compared to the baseline uniform coarse-grained structure), while maintaining an acceptable total elongation of 8.4%. The impact of cryo-pre-straining on the microstructure and mechanical properties of the MEA was assessed using in-situ synchrotron X-ray diffraction analysis. Cryo-pre-straining (36%) achieved a higher dislocation density (6.1 × 1015 m−2) compared to room-temperature straining (2.5 × 1015 m−2). The stress contribution from HCP-martensite and the evolution of dislocation density during loading were quantified, along with observations of negative stacking fault probability and strain-induced HCP→FCC reverse transformation in cryo-pre-strained samples under loading conditions. Furthermore, the contributions of regulated microstructures to the enhancement of yield strength were quantitatively assessed.