To investigate the level of serum S100A6 in patients with bladder cancer and in healthy controls and compare these levels with clinicopathologic findings, we evaluated the level of serum S100A6 in 30 healthy controls and 50 patients with bladder cancer diagnosed via transurethral resection of bladder tumor and/or radical cystectomy. S100A6 in sera was detected by employing automatic dot blot systems, and the micro Dot Blot array with a 256-solid pin configuration. The normalized signal of serum S100A6 expression in bladder cancer patients was significantly higher than that of healthy controls (P = 0.001). Serum S100A6 expression of non-muscle-invasive cancer (NMIC) was significantly higher than that of healthy controls (P = 0.04). Furthermore, the S100A6 serum level in patients with muscle-invasive bladder cancer was significantly higher than that in patient with NMIC (P = 0.004). The sensitivity and specificity were 48.0% (95% CI: 0.337-0.626) and 93.3% (95% CI: 0.779-0.992), respectively. The area under the curve was 0.727. Serum S100A6 expression is a potentially effective detection marker for bladder cancer. Applying this serum marker to clinical practice would require less-invasive examinations of patients and would help to detect life-threatening cancerous lesions earlier than current modalities.