Background Linezolid has shown strong antimicrobial activity against multidrug-resistant (MDR)/rifampin-resistant strains of Mycobacterium tuberculosis. Linezolid achieves clinical efficacy mainly through area under the concentration time curve/minimum inhibitory concentration ratio in the infected lesion site. Previous studies mainly focused on the relationship between linezolid concentrations in the blood and infected bone tissue when the blood drug concentration reached the peak 2 h after administration. However, we do not know whether linezolid can maintain the same bone/plasma ratio in infected bone tissue when the blood concentration reaches the trough level. Therefore, this study aimed to evaluate the penetrability of linezolid into bone tissue 24 h after administration in patients with MDR spinal tuberculosis (TB). Methods Nine MDR spinal TB patients, who received a treatment regimen including linezolid and underwent surgery, were enrolled prospectively from April 2017 to March 2019. Blood and diseased bone tissue specimens were collected simultaneously during operations 24 h after taking 600 mg of linezolid orally. Linezolid concentrations in plasma and diseased bone tissue specimens were determined by high-performance liquid chromatography tandem mass spectrometry. Results Following a 600 mg oral administration of linezolid 24 h before surgery, median concentrations of linezolid in plasma and diseased bone tissue for the 9 patients were 1.98 mg/L (range 0.30-3.44 mg/L) and 0.60 mg/L (range 0.18-2.13 mg/L), respectively, at resection time. The median diseased bone/plasma linezolid concentration ratio was 0.48 (range 0.30 0.67). Pearson's correlation analysis showed that linezolid concentrations in the plasma were positively related to those in diseased bone tissue (r = 0.949, p < 0.001). Conclusions After 24 h of medication, linezolid still had good penetrability into diseased bone tissue in patients with MDR spinal TB.