The overexpression of β-tubulin III (TUBB3) in tumor tissues was reversely related with the efficacy of paclitaxel and clinical outcome in different cancers. In this study, we aimed to investigate the association between serum levels of TUBB3 and clinical outcome in advanced gastric cancer patients receiving first-line paclitaxel plus capecitabine. One hundred and twenty-eight advanced gastric cancer patients receiving first-line paclitaxel plus capecitabine in Peking University Cancer Hospital from December 2006 to October 2010 were enrolled in the study. Serum samples from 32 healthy individuals were used as controls. TUBB3 expression level in advanced gastric cancer was significantly higher than that in healthy control group (31.6 ± 17.8 ng/mL vs. 16.9 ± 3.8 ng/mL, p < 0.001). For all patients, the clinical benefit rate (CBR), median progression-free survival (PFS), and overall survival (OS) were 55.6 %, 179 and 306 days, respectively. The CBR, median PFS, and OS in patients with low (n = 27) and high levels (n = 101) of TUBB3 were 95.8 %/45.1 % (low vs. high, p < 0.001), 190 days/166 days (p = 0.064), and 360 days/297 days (p = 0.023), respectively. Cox multivariate regression analysis demonstrated that the serum levels of TUBB3 were an independent prognostic factor for advanced gastric cancer patients (HR = 1.950; 95 % CI, 1.242-3.062; p = 0.004). This study indicated that low levels of TUBB3 in serum could predict better response and survival for advanced gastric cancer patients receiving paclitaxel plus capecitabine, which could be used to select patients who would benefit from this regimen.