To develop a diagnosis system for detection pepper mild mottle virus (PMMoV) via molecular hybridization, total nucleic acid was extracted with modified CTAB method from pepper leaves infected by PMMoV, and the specific fragment was amplified using RT–PCR technology. PMMoV was detected by adopted the plasmid DNA from specific fragment of PMMoV as template, and the PMMoV-cDNA labeled with digoxigenin prepared via PCR as probe. The results indicated that: 1) PMMoV could be even detected from the total nucleic both diluted to 80-fold (amount to 12.5 μg leaves) for dry leaves and 640-fold (amount to 1.6 μg leaves) for fresh leaves. Easy conservation and long distance transport for dry leaves were advantageous to perform centralized detection for collected samples in bulk; 2) nucleic acid extracted using the rapid method from pepper leaves could be diluted to 80-fold (amount to 12.5 μg leaves) for PMMoV detection. The diagnostic system could meet the need of routine detection of PMMoV.