Hydrogel scaffolds are attractive for tissue defect repair and reorganization because of their human tissue-like characteristics. However, most hydrogels offer limited cell growth and tissue formation ability due to their submicron- or nano-sized gel networks, which restrict the supply of oxygen, nutrients and inhibit the proliferation and differentiation of encapsulated cells. In recent years, 3D printed hydrogels have shown great potential to overcome this problem by introducing macro-pores within scaffolds. In this study, we fabricated a macroporous hydrogel scaffold through horseradish peroxidase (HRP)-mediated crosslinking of silk fibroin (SF) and tyramine-substituted gelatin (GT) by extrusion-based low-temperature 3D printing. Through physicochemical characterization, we found that this hydrogel has excellent structural stability, suitable mechanical properties, and an adjustable degradation rate, thus satisfying the requirements for cartilage reconstruction. Cell suspension and aggregate seeding methods were developed to assess the inoculation efficiency of the hydrogel. Moreover, the chondrogenic differentiation of stem cells was explored. Stem cells in the hydrogel differentiated into hyaline cartilage when the cell aggregate seeding method was used and into fibrocartilage when the cell suspension was used. Finally, the effect of the hydrogel and stem cells were investigated in a rabbit cartilage defect model. After implantation for 12 and 16 weeks, histological evaluation of the sections was performed. We found that the enzymatic cross-linked and methanol treatment SF5GT15 hydrogel combined with cell aggregates promoted articular cartilage regeneration. In summary, this 3D printed macroporous SF-GT hydrogel combined with stem cell aggregates possesses excellent potential for application in cartilage tissue repair and regeneration.