Objective To establish a real-time fluorescent PCR assay for detecting Botryosphaeria stevensii, which is the causal agent of Diplodia canker on apple and is presently subjected to phytosanitary legislation in China.Method A pair of specific primers and a TaqMan MGB probe were designed based on the conserved sequences of β-tubulin genes of B. stevensii and related species. The specificity and sensitivity of the probe were evaluated using DNAs of B. stevensii strains and recombinant plasmid of β-tubulin gene sequence as positive standard, respectively.Result The probe of BsP267 displayed specificity to B. stevensii strains with positive amplification, while there was no crossing reaction with related species and other common fruit rot pathogens. The PCR amplification efficiency was 105.858% and the detection sensitivity of DNA reached 1 fg/μL.Conclusion The real-time PCR method developed in this study can detect B. stevensii from apple with strong specificity and high sensitivity, thus can be used for the prevention, control, detection and quarantine of this disease.