This study explores the development of artificial spider silk with properties similar to native silk using a microfluidic device. The strategy involves ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of β-sheet formation. Key findings include: 1. **Fiber Formation**: A threshold shear stress of approximately 72 Pa is required for fiber formation, and β-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. 2. **β-Sheet Content**: The MaSp2 fiber formed has a β-sheet content (29.2%) comparable to that of native dragline silk, with a shear stress requirement of 111 Pa. 3. **Influence of Polyalanine Blocks**: These blocks have limited influence on liquid-liquid phase separation and hierarchical structure. 4. **Shear-Induced Crystallization**: The microfluidic system successfully induces rapid self-assembly of recombinant MaSp2 into hierarchically structured fibers, with tunable β-sheet abundance and at near-instantaneous speeds. 5. **Computational Analysis**: Numerical simulations reveal the distribution of components and interplay of physical forces during fiber assembly, highlighting the critical role of shear stress in β-sheet formation. The study provides insights into the shear-induced crystallization and sequence-structure relationship of spider silk, offering significant implications for the rational design of artificially spun fibers.This study explores the development of artificial spider silk with properties similar to native silk using a microfluidic device. The strategy involves ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of β-sheet formation. Key findings include: 1. **Fiber Formation**: A threshold shear stress of approximately 72 Pa is required for fiber formation, and β-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. 2. **β-Sheet Content**: The MaSp2 fiber formed has a β-sheet content (29.2%) comparable to that of native dragline silk, with a shear stress requirement of 111 Pa. 3. **Influence of Polyalanine Blocks**: These blocks have limited influence on liquid-liquid phase separation and hierarchical structure. 4. **Shear-Induced Crystallization**: The microfluidic system successfully induces rapid self-assembly of recombinant MaSp2 into hierarchically structured fibers, with tunable β-sheet abundance and at near-instantaneous speeds. 5. **Computational Analysis**: Numerical simulations reveal the distribution of components and interplay of physical forces during fiber assembly, highlighting the critical role of shear stress in β-sheet formation. The study provides insights into the shear-induced crystallization and sequence-structure relationship of spider silk, offering significant implications for the rational design of artificially spun fibers.