To compare the clinical outcomes and biomechanical characteristics of 1-, 2-, and 3-level pedicle subtraction osteotomy (PSO), and establish selection criteria based on preoperative radiographic parameters. Patients undergone PSO to treat ankylosing spondylitis from February 2009 to May 2019 in Sun Yat-sen Memorial Hospital of Sun Yat-sen University were enrolled. According to the quantity of osteotomy performed, the participants were divided into group A (1-level PSO, n = 24), group B (2-level PSO, n = 19), and group C (3-level PSO, n = 11). Clinical outcomes were assessed before surgery and at the final follow-up. Comparisons of the radiographic parameters and quality-of-life indicators were performed among and within these groups, and the selection criteria were established by regression. Finite element analysis was conducted to compare the biomechanical characteristics of the spine treated with different quantity of osteotomies under different working conditions. Three-level PSO improved the sagittal parameters more significantly, but resulted in longer operative time and greater blood loss (p < 0.05). Greater stress was found in the proximal screws and proximal junction area of the vertebra in the model simulating 1-level PSO. Larger stress of screws and vertebra was observed at the distal end in the model simulating 3-level PSO. Multilevel PSO works better for larger deformity correction than single-level PSO by allowing greater sagittal parameter correction and obtaining a better distribution of stress in the hardware construct, although with longer operation time and greater blood loss. Three-level osteotomy is recommended for the patients with preoperative of global kyphosis > 85.95°, T1 pelvic angle > 62.3°, sagittal vertical alignment > 299.55 mm, and pelvic tilt+ chin-brow vertical angle > 109.6°.