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The <i>Agrobacterium</i> tumefaciens-mediated transformation for blueberries remains less efficient than is desirable. A new leaf callus regeneration and genetic transformation system was investigated in blueberries in this study. The leaf explants of <i>cv.</i> ‘Legacy’ and ‘Northland’ were used to establish the stable callus induction system when placed on the woody plant medium (WPM) supplemented with 1.0 mg·L⁻¹ 2, 4-D, 0.4 mg·L⁻¹ 6-BA for 30 d; then, the callus was sub-cultured in the proliferation medium supplemented with 1.5 mg·L⁻¹ 2, 4-D, 0.4 mg·L⁻¹ 6-BA in the darkness at 25 °C every 30 days. The co-cultivation of callus with <i>A. tumefaciens</i> was operated on WPM plus 100 μM acetosyringone for 4 days; then, the transferred callus was grown in WPM supplemented with 1.5 mg·L⁻¹ 2,4-D, 0.4 mg·L⁻¹ 6-BA, 50 mg·L⁻¹ hygromycin, and 200 mg·L⁻¹ cefotaxime. The <i>VcCHS</i> transgenic blueberry callus with both GFP signal and Hyg resistance was obtained from the transformed callus of <i>cv.</i> ‘Northland’. The rate of GFP signal detected in the transformed callus was as high as 49.02%, which was consistent with the PCR assay. Collectively, this study provides a highly efficient genetic transformation system in blueberry callus and a powerful approach for the molecular breeding of blueberries.