Pathogen causing spruce needle blight was isolated from the artificial spruce forest on Erlang Moutain, and the amplification sequence from the pathogen using fungus general primers ITS1 and ITS4 showed 99% similarity with the Rhizosphaera kalkhoffii in NCBI. Internal transcribed spacer (ITS) sequences of the isolated pathogen and Rhizosphaera kalkhoffii (R. kalkhoffii) in NCBI were compared and analyzed, and 20 pairs of primers were designed with softwareprimer5.0. The amplification results for 32 strains combined with sequencing results showed that 3 primer pairs ZYZ6, ZYZ7 and ZYZ8 specific for R. kalkhoffii were obtained. The sensitivity of the 3 specific primers was tested by changing their concentration. The results showed primer pair ZYZ6F/ZYZ6R had the highest sensitivity which can detect the pathogen at genomic DNA concentration of 0.1 pg/μL in 25 μL PCR reaction system. Primer pair ZYZ6F/ZYZ6R was used to detect the pathogen from the DNA of the plant leaves of spruce. Sequencing results showed the sequence of the 310 bp amplified bands showed 99% similarity with the recorded sequence of strain Rhizosphaera kalkhoffii in GenBank.