Abstract: At present, the existing scaffold materials cannot well meet the high strength and toughness requirements for the repairing of typical stressed tissues. And the commercial software for 3D printing scaffold modeling and mechanical performance simulation is complex to operate and has poor interoperability with 3D printers. In order to solve these problems, a silk fibroin (SF) based bio-ink was prepared for tough scaffold, and a software was developed for compression performance simulation of 3D printing scaffold based on SF and finite element analysis strategy. The printability of the bio-ink and the mechanical properties of the corresponding hydrogels and 3D printing scaffolds were characterized. The cell compatibility of the related scaffolds was evaluated by using fibroblasts and living/dead staining kit. Based on the SF based bio-ink, scaffolds with different heights and porosities were designed and printed. By using the developed software and the universal testing machine, the compression performance of related scaffolds was simulated and tested separately, and further compared with each other. Results show that the SF based bio-ink has good printability. And the 3D printing scaffold prepared by this ink presents high strength and toughness (modulus of elasticity: (1.86 ± 0.28)MPa, modulus of compression: (1.95 ± 0.11)MPa, elongation at break: (114.03 ± 14.40)%, compression strain ≥ 70%). In addition, the scaffold has good cell compatibility (L929 cell viability≥92.4%), which is expected to be used for the repair and regeneration of stressed tissues. The software is easy to operate, and can be used not only for the modeling of 3D printing scaffolds, but also for accurately predicting their compressive mechanical properties. In addition, the model data can also be directly imported into the 3D printer to guide the efficient preparation of the corresponding 3D printing scaffolds. This research is expected to provide important guidance for the rapid design and fabrication of tissue specific 3D printing scaffolds with bionic structure and mechanical properties.