The effects of the hormone combination of cytokinin ZT, TDZ, auxin IBA, NAA and dark culture time on the establishment of regeneration system of in vitro leaves of "Spartan" blueberry were studied. The results showed that when TDZ was 0.02 mg/L and IBA was 0.3 mg/L, the average number of buds induced was 4.61, which was the highest in all treatments, and the time of bud differentiation was fast, and the density of bud cluster was large; the highest callus induction rate was 100% in dark culture for 20 d, and the largest callus. Comprehensive analysis showed that the suitable medium for leaf differentiation of "Spartan" blueberry was WPM + TDZ 0.02 mg/L + IBA 0.3 mg/L, and the suitable dark culture time for leaf callus induction of "Spartan" blueberry was 20 d.