The construction of segregated structures within conductive polymer composites (CPCs) is a promising strategy for enhancing the effectiveness of electromagnetic interference (EMI) shielding. Herein, three-dimensional (3D) printed carbon fiber powder (CF)/polylactic acid (PLA) composites were prepared with various filling ratio, filling pattern, thickness and CF contents. The results showed that CF was uniformly dispersed in the PLA. Additionally, the modulus of the printed CF/PLA composite displayed an improvement in comparation to injected composite, despite a decrease of the strength. Within the final printed CF/PLA composite, CF integrated into 3D printed PLA formed an interconnected conductive network based on 3D printing technology. The performances of 3D printed CF/PLA composite exhibited superior comprehensive performance when the filling ratio was 50 %, combined with a grid filling pattern, a thickness of 5 mm, and CF content of 20 %, and its shielding efficiency could reach 99.67 %. Meanwhile, the EMI shielding mechanism of 3D printed CF/PLA composite was dominantly of absorption loss. This work suggests that 3D printing technology provides a simple and effective method for developing 3D printed CF/PLA composite with excellent EMI performance, suitable for different electronic devices and radiation source fields.