Mass spectrometry is a central technique in glycomic analysis. However, there is no generic software tool for automated, confident analysis of tandem-mass spectrometry based glycomic data. Here, we propose GlycoNote – a generic and reliable search engine for tandem-mass spectrometry based glycomics. A false discovery rate analysis based on iterative decoy searching was specifically designed for glycomic data analysis. We apply GlycoNote to the analyses of distinct glycomic samples, including human milk oligosaccharides, N/O-glycome from human cell line and polysaccharides from plant. To further demonstrate the general utility of GlycoNote, automated analyses of nonnative glycomes (N-glycome labeled with aniline and permethylated N-glycome) or atypical glycans (O-glycome with N-acetylneuraminic acid / N-glycome from C. elegans) were performed. More importantly, an open-search mode was introduced for the elucidation of component heterogeneity in samples. GlycoNote could be an important tool in the rapidly growing efforts toward comprehensive glycomic analysis.