Capillary-force-driven self-assembly (CFSA) has been combined with many top-down fabrication methods to be alternatives to conventional single micro/nano manufacturing techniques for constructing complicated micro/nanostructures. However, most CFSA structures are fabricated on a rigid substrate, and little attention is paid to the tuning of CFSA, which means that the pattern of structures cannot be regulated once they are manufactured. Here, by combining femtosecond laser direct writing with CFSA, a flexible method is proposed to fabricate self-assembled hierarchical structures on a soft substrate. Then, the tuning of the self-assembly process is realized with a mechanical-stretching strategy. With this method, different patterns of tunable self-assembled structures are obtained before tuning and after release, which is difficult to achieve with other techniques. In addition, as a proof-of-concept application, this mechanical tunable self-assembly of microstructures on a soft substrate is used for smart displays and versatile micro-object trapping.