Introduction A trade-off between successful surgery and minimizing the operation delay for patients with spinal tuberculosis (TB) is a major consideration to determine the duration of preoperational anti-TB treatment (AAT). In this study, 2 and 4 weeks preoperative AAT durations were compared for their influence on the operation outcomes. Method A multicenter, prospective, randomized trial was conducted in four hospitals in China. New patients with spinal TB were recruited and randomly allocated to two groups (2 or 4 weeks' preoperative treatment) and administered the standardized first-line anti-TB drugs. The symptom changing and indicators reflecting recovery and side effects of the treatment were monitored. Patient was followed up for another 18 months after completion of treatment. Results In total, 150 eligible patients were enrolled between June 2014 and December 2016, and 13 patients were excluded after the enrollment. The remaining 137 participants were randomly allocated to the 2-week group (n = 68) or the 4-week group (n = 69). These two groups acquired similar surgical outcomes, considering wound healing rate within 3 months after the operation (94.20%, 65/69 vs 89.71%, 61/68; P = 0.333) and bony fusion rate within 6 months (98.46%, 64/65 vs 95.45%, 63/66; P = 0.317). However, the culture positive rate of pus collected during operation in the 4-week group (41.94%) was significantly lower than that of the 2-week group (60.94%, P = 0.033). No reoccurrence of disease was observed in either group during the 18-month follow-up period. Conclusion Patients with spinal TB administered 2 or 4 weeks of preoperative anti-TB treatment acquired similar surgical outcomes. However, patients who underwent the operation sooner suffered 2 weeks less agony from the disease.