It is of great value to sieve suitable nutrient medium in tissue culture of blueberry for its special requirement on growing acid soil and for solving issues of slow growth, rooting difficulty, as well as high cost of using growth regulatory substance (e.g. zeatin). Box–Behnken design was adopted to explore the effect of wood vinegar on blueberry growth by adding them into rooting medium and transplanting medium in tissue culture. The results indicated that although wood vinegar had an inhibited effect on the callus formation of leaves and stem, it could accelerate propagation of stem. Woody plant medium (WPM +0.5 mg/L 6–BA+1.0 mg/L ZT) was the best basic medium for tissue culture of blueberry. Wood vinegar had significant effects on the propagation of blueberry stem, and little interaction between zeatin and 6-BA on proliferation. The selected model was fitted well with actual test, and it could explain 96.5% of total response value. Wood vinegar (1/2 WPM+1.0 mg/L NAA+0.5 mg/L IBA+10 mL/L wood vinegar) was a good medium which could increase 66% rooting rate compared with contrast group. In addition, wood vinegar could greatly increase survival rate of transplanting in that an addition of 20 mL/kg wood vinegar could improve survival rate to 93.3% with robust seedling, dense green stem and leaf, as well as the high stem which could reach 8.1 cm. The addition of wood vinegar could reduce the amount of zeatin use, for it could cut reagent cost down to 70%. Wood vinegar can be applied in blueberry tissue culture as a kind of plant growth regulator.