The precise fabrication and controllable actuation of magnetic microspheres hold significant application value in biomedicine, microfluidic chips and other fields. Based on femtosecond laser two-photon polymerization technology (FLTPP), two methods are adopted to prepare magnetic microspheres in this study. Magnetic microspheres are fabricated via photoresist modification and post-treatment processes. Meanwhile, a 3D magnetic actuation system composed of a three-axis movable magnetic drive module and a real-time imaging system is constructed, enabling the flexible 3D actuation and real-time dynamic monitoring and visualized observation of magnetic microspheres. The results demonstrate that the magnetic microspheres exhibit sensitive magnetic response characteristics. The constructed magnetic actuation system features large travel range (XY: ±6.5 mm, Z: 10 mm), high precision (20 μm) and flexible manipulation, enabling stable locomotion of the microrobots in straight channels, L-shaped channels, and square channels. This study provides a technical reference for the fabrication and manipulation of magnetic micro/nano devices, and lays a foundation for their subsequent integrated applications in microfluidic systems.