Banana fusarium wilt,caused by Fusarium oxysporum f. sp.cubense race 4 (Foc4), is one of the most destructive diseases of banana worldwide. To obtain the toxin of Foc4 effectively and provide the foundation for further research, the culture filtrates of Foc 4 obtained from different culture media,in different incubation modes, with different incubation time, incubation temperatures, initial pH values of Richard medium, rotation speeds of shaker, and inoculation amounts, under different illumination conditions were tested for their bioactivities to banana plantlets in vivo by using root-dipping method. An optimized system for toxin production of Foc 4 was established, in which the optimal condition for Foc 4 to produce toxin was obtained when it was cultured in Richard medium with an initial pH 7-9 and 5 mycelial plugs per 300 mL,in an incubation temperatures of 25-30 ℃ under continuous illumination for 24 h in a shaker at 140 r/min for 9-12 days.