Background: Globally, colorectal cancer (CRC) is the fourth most frequently occurring cancer and is the second leading cause of cancer-related deaths. Early detection and diagnosis of pre-cancer lesions and CRCs at curable stages could prevent the disease or improve patient survival. Colonoscopy, the current gold-standard approach for CRC screening, is an invasive procedure, and novel non-invasive alternatives are needed. Methods: In present study, we developed a novel multi-target fecal DNA based assay, which integrates detection of two stool DNA (sDNA) methylation markers by quantitative methylation-specific PCR (qMSP) and immunochemical fecal occult blood test. We performed a multi-center case-control study to validate the diagnostic performance of this novel multi-target assay. Thus far, we collected stool specimens from a total of 784 subjects including CRC, advanced adenoma, nonadvanced neoplasms and individuals with negative findings on colonoscopy from six sites across China. All participants underwent colonoscopy; the stool specimens were tested with the multi-target fecal DNA test as well as a quantitative FIT test (OC FIT-CHEK, Eiken) in parallel in order to perform head-to-head comparison of the test performance of both assays. Results: The multi-target fecal DNA test demonstrated a higher sensitivity (96.1%) than FIT (85.3%) for CRCs (n=204). Meanwhile, 20 of 39 (51.3%) patients with advanced adenoma tested positive by the fecal DNA test, which was remarkably superior to FIT (7 of 39 tested positive, 17.9%). Among 541 participants with nonadvanced neoplasms or negative findings on colonoscopy, the specificity of the DNA test was 86.9%, compared with a specificity of 93.1% achieved by FIT. Within the control group, the DNA test and FIT yielded a specificity of 90.5% and 94.9% respectively for individuals with negative results on colonoscopy. Conclusions: The novel multi-target fecal DNA test exhibited notably superior sensitivity for both CRC and advanced adenoma cases than the commercially quantitative FIT test, while sacrificing some specificity. Our results suggest that this novel multi-target fecal based test may be used as an effective tool for early detection and pre-screening of CRC. Prospective study will be conducted to further evaluate the clinical performance and cost-effectiveness of this assay.