[Aim]  To establish a standardized method for cultivating germ-free strains of Bactrocera dorsalis, providing robust technical support for investigating the interactions between gut microbiota and host. [Methods]  The operational procedure comprised four key steps: Environmental control, material sterilization, embryo surface sterilization, and axenic cultivation. The effectiveness of the axenic rearing was comprehensively evaluated using plate culture combined with PCR amplification and electrophoretic detection of bacterial 16S rDNA and fungal ITS1 regions. [Results]  Following the above procedure, surface-sterilized embryos consistently developed to the pupal stage. Both plate culture and molecular detection (PCR amplification and electrophoresis of 16S rDNA and ITS1 regions) yielded consistent results, indicating no detectable exogenous microbial contamination inside or outside the obtained larvae. [Conclusion]  By optimizing operational procedures and environmental controls, this study established a stable and reproducible system for cultivating axenic strain of B. dorsalis, providing a standardized experimental model for functional studies on gut symbionts and other aspects.