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Chalcone synthase (CHS) is the first key enzyme-catalyzing plant flavonoid biosynthesis. Until now, however, the blueberry CHS gene family has not been systematically characterized and studied. In this study, we identified 22 <i>CHS</i> genes that could be further classified into four subfamilies from the highbush blueberry (<i>Vaccinium corymbosum</i>) genome. This classification was well supported by the high nucleotide and protein sequence similarities and similar gene structure and conserved motifs among VcCHS members from the same subfamily. Gene duplication analysis revealed that the expansion of the blueberry CHS gene family was mainly caused by segmental duplications. Promoter analysis revealed that the promoter regions of <i>VcCHSs</i> contained numerous <i>cis</i>-acting elements responsive to light, phytohormone and stress, along with binding sites for 36 different types of transcription factors. Gene expression analysis revealed that Subfamily I <i>VcCHSs</i> highly expressed in fruits at late ripening stages. Through transient overexpression, we found that three <i>VcCHSs</i> (<i>VcCHS13</i> from subfamily II; <i>VcCHS8</i> and <i>VcCHS21</i> from subfamily I) could significantly enhance the anthocyanin accumulation and up-regulate the expression of flavonoid biosynthetic structural genes in blueberry leaves and apple fruits. Notably, the promoting effect of the Subfamily I member <i>VcCHS21</i> was the best. The promoter of <i>VcCHS21</i> contains a G-box (CACGTG) and an E-box sequence, as well as a bHLH binding site. A yeast one hybridization (Y1H) assay revealed that three anthocyanin biosynthesis regulatory bHLHs (VcAN1, VcbHLH1-1 and VcbHLH1-2) could specifically bind to the G-box sequence (CACGTG) in the <i>VcCHS21</i> promoter, indicating that the expression of <i>VcCHS21</i> was regulated by bHLHs. Our study will be helpful for understanding the characteristics and functions of blueberry <i>CHSs.</i>