HPLC-ESI-MS/MS was applied to rapid analysis of flavonoids in the extract of blueberry. Anthocyanins and flavonols were identified as the primary components. The different structures lead to the differences in their UV-Vis absorptions, ionizations and fragmentations. Although both shown absorption maximum at 270 nm, anthocyanins and flavonols possessed characteristic absorption at 530 and 372 nm, respectively. Anthocyanins and flavonols could be distinguished since anion and radical anion were generated from flavonols in the negative ion ESI-MS, while anthocyanins gave no obvious signal under the same conditions. Different characteristic product ions in MS/MS diagram further confirmed the distinctions in structure between anthocyanins and flavonols. This study is helpful for the quality analysis and control of the extract of blueberry.