To explore and improve the utilization efficiency of Gentiana macrophylla resources.The germination of Gentiona macrophylla seeds were used as explants to induce calluses; Cultured on various culture media and condition for callus induction and subculture by test; The content of gentiopicroside in callus was determined by HPLC.The optimum medium for callus was MS +2,4-D (1.0 mg/L) + 6-BA (0.5 mg/L), and MS +2,4-D (1.0 mg/L) +6-BA (0.5 mg/L) + NAA (0.5 mg/L) was the optimum for subculture,the optimum time of subculture was 30 d; The accumulated gentiopicroside reached the highest level with 0.78 mg/g DW when entered 28-30 d.The optimized culture method of callus of Gentiana. macrophylla is obtained; The growth curve of callus of Gentiona macrophylla turns on "S" appearance and the accumulated gentiopicroside increases along with the callus appreciation.