Abstract: Facing diverse mission requirements such as deep space exploration, high-precision Earth observation, and space-based solar power stations, ultra-large space structures have become crucial equipment for strategic objectives including space resource utilization and the exploration of the universe. However, the mechanical behavior of the space structures in the process of on-orbit assembly is highly dependent on the assembly sequence, making the optimization of “progressive growth” structures a particularly challenging task. To overcome this limitation, this study introduces a bio-inspired sequential growth topology optimization approach for on-orbit assembly, incorporating seed initialization, neighborhood definition, growth criteria, and dynamic design domain updating. A compliance-minimization sequential growth optimization model is established to capture the stepwise construction process of on-orbit structures. Systematic investigations of the hexagonal assembly structures reveal that growth speed significantly affects both topology and mechanical performance. Meanwhile, a frequency-maximization sequential growth optimization model is also established, revealing growth patterns driven by dynamic performance and the influence of sensitivity filter radii. Results reveal that larger filter radius leads to overly smoothed growth paths with inferior performance compared with the relatively localized growth paths associated with smaller radius. Overall, the bio-inspired sequential growth topology optimization method effectively reflects the progressive assembly characteristics of on-orbit structures and demonstrates promising engineering applicability in both static and dynamic performance optimization.