The stem segments of tissue culture seedlings of Lonicera praeflorens Batal were adopted as the materials in the experiment. And the Box-Behnken Response Surface Methodology was used to optimize the subculture conditions of Lonicera praeflorens Batal in order to provide reference for establishing the rapid proliferation system of the in vitro tissue culture of Lonicera praeflorens Batal. The addition range of hormone was determined by single factor test method, and the optimized culture medium for proliferation was obtained by Response Surface Methodology accordingly. The results showed that: the descending order of the influence that different hormone sorts had on the stem segments subculture of tissue culture seedlings of Lonicera praeflorens Batal is: 6-BA＞IAA＞IBA. Furthermore, the multiplication coefficient of the subculture of stem segments was 7.66, under the condition of 1.9 mg/L 6-BA, 0.76 mg/L IBA, and 0.27 mg/L IAA, which are very close to the predicted values.