Viable but non-culturable Salmonella was detected by RT-PCR with primers designed according to the differential viable but non-culturable (VBNC) genes, ybbB and rh1B. The results showed that primers C1 and C5 respectively amplified fragments about 198 and 137 bp, their nucleotide sequence homology with ybbB and rh1B was both over 99%, and the electrophoretogram form VBNC Salmonella was different from that of normal Salmonella, Vibrio cholerae, et al. The lowest cDNA detection level with primers C1 and C5 in VBNC Salmonella was respectively 30.65 and 3.065 pg/μL. Primers C1 and C5 with specificity and sensibility were applied to establish the detection method in VBNC Salmonella.