[Objective] To study the factors influencing the callus induction of Polygala tenuifolia Willd., so as to establish callus culture method. [Method] The tissue culture method was adopted to compare the effects of different explants, pH, temperature, sunlight, hormone types and proportions on callus induction of Polygala tenuifolia Willd. [Result] The induction rate of cotyledon on MS was the highest at 88%; the callus induced from leaves and stems were few; the effect of different pH on the callus growth was not obvious; the optimum temperature for callus induction was 28 ℃; the induction rate under 66% light conditions was higher than that under 33% light conditions; the induction rate under the hormone combination of 1.0 mg/L 6-BA + 0.1 mg/L NAA was the highest. [Conclusion] The optimum culture conditions for the callus of Polygala tenuifolia Willd. were MS + 1.0 mg/L 6-BA + 0.1 mg/L NAA, pH 6.8, culture temperature 28 ℃, and the alternation between 66% light culture for 18 h and dark culture for 6 h. [Objective] To study the factors influencing the callus induction of Polygala tenuifolia Willd., So as to establish callus culture method. [Method] The tissue culture method was adopted to compare the effects of different explants, pH, temperature, sunlight, hormone types and proportions on callus induction of Polygala tenuifolia Willd. [Result] The induction rate of cotyledon on MS was the highest at 88%; the callus induced from leaves and stems few; the effect of different pH on the callus growth was not obvious; the optimum temperature for callus induction was 28 ° C; the induction rate under 66% light conditions was higher than that under 33% light conditions; the induction rate under the hormone combination of 1.0 mg / L 6-BA + 0.1 mg / L NAA was the highest culture condition for the callus of Polygala tenuifolia Willd. were MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA, pH 6.8, culture temperature 28 ° C, and the alternation between 66% light culture for 18 h and dark culture for 6 h.