In recent years, different detection and isolation ways have emerged in the era of breast cancer. Despite the clinical significance of CTCs, the absence of unbiased and easy separation techniques poses a major obstacle to the integration of CTCs into clinical applications. This study presents an inertial microfluidic chip with a trapezoidal cross-section for rapid, simple, and viable separation of circulating tumor cells (CTCs) of MCF-7 cell line from breast cancer patient blood by using an optimized additive manufacturing (AM) technique. This microchannel was fabricated by SLA (stereolithography apparatus) 3D printer. Also, we demonstrated the influence of slant angle in trapezoidal cross-section in the formation of Dean vortices and suitable flow rate for separating by simulation and experiments. In this regard, by increasing the slant angle, the proper flow rate for this study was obtained at 1.2 mL/min in COMSOL simulating, and spiking CTCs to Phosphate-Buffered Saline (PBS) confirmed it (recovery rate was 88 ± 3.5%). In the next step, CTCs were spiked to 7.5 mL lysed healthy blood sample. White blood cells (WBCs) and CTCs were isolated in this stage. The recovery rate of this test was 86 ± 2.9%. The results confirmed that the shears and stresses applied to the cells during sample processing did not compromise their viability, and more than 90% of the cells were recovered alive. In the last step, the trapezoidal microchannel was used in clinically detecting five breast cancer patients with different stages. The outcomes showed that the chip had high sensitivity in detecting CTCs from blood samples of 4 of 5 patients (80%) (Range 6–21 CTCs/mL blood sample) by immunocytochemistry (ICC) technique. We anticipate that this straightforward inertial microfluidic approach can overcome the limitations associated with traditional affinity-based methods for CTC separation. It has the potential to facilitate fundamental research on CTCs, aiding in treatment guidance and improving patient outcomes.