Overexpressions of synuclein-gamma (SNCG) in different cancers display stage-specific patterns. At present, appropriate anti-SNCG monoclonal antibodies (mAbs) with high specificity and affinity are unavailable for different immunoassays in clinical applications. In this study, we generated 10 mAbs against endogenous SNCG and evaluated SNCG levels in several colorectal cancer cell lines, serum samples and tumor tissues from colorectal cancer (CRC) patients. Elevated SNCG levels in cancer cell lines evaluated by a novel sandwich ELISA were consistent with data obtained from Western blot. Secreted SNCG protein levels in sera from CRC patients could be detected by the sandwich ELISA and were further confirmed by Western blot analysis following SNCG enrichment. Immunohistochemical results showed that SNCG was highly expressed in tumor cells of CRC patients, but was undetectable in the adjacent normal epithelium. Taken together, these novel anti-SNCG mAbs specifically recognized endogenous SNCG and were suitable for measuring SNCG levels in cell lysates, human serum samples, and tumor tissues. Elevated serum SNCG and overexpressed SNCG in tumor tissue from CRC patients suggest SNCG is a potential biomarker for CRC.