Mechanical signals influence the morphology, function, differentiation, proliferation, and growth of cells. Due to the small size of cells, it is essential to analyze their mechanobiological responses with an in vitro mechanical loading device. Cells are cultured on an elastic silicone membrane substrate, and mechanical signals are transmitted to the cells by the substrate applying mechanical loads. However, large areas of non-uniform strain fields are generated on the elastic membrane, affecting the experiment’s accuracy. In the study, finite-element analysis served as the basis of optimization, with uniform strain as the objective. The thickness of the basement membrane and loading constraints were parametrically adjusted. Through finite-element cycle iteration, the “M” profile basement membrane structure of the culture chamber was obtained to enhance the uniform strain field of the membrane. The optimized strain field of culture chamber was confirmed by three-dimensional digital image correlation (3D-DIC) technology. The results showed that the optimized chamber improved the strain uniformity factor. The uniform strain area proportion of the new chamber reached 90%, compared to approximately 70% of the current chambers. The new chamber further improved the uniformity and accuracy of the strain, demonstrating promising application prospects.