Polymerase chain reaction (PCR) method was developed for the specific detection of insect-resistant rice containing cry1Ac gene derived from Bacillus thuringiensis (Bt). Primers were designed from the 35S promoter, NOS terminator, cry1Ac gene, and sucrose phosphate synthase (SPS) for general screening of Bt rice. By sequencing the PCR products from the two putative kinds of Bt rice, we designed a specific primer from the junction region between the cry1Ac gene and the NOS terminator that had been inserted into Bt rice. The construct-specific primer was employed to amplify a 147 bp product in the two lines of Bt rice. No amplified products were observed from the other Bt crops with various Bt genes introduced. In qualitative PCR analysis, the limit of detection was 0.005 ng from genomic DNA of Bt rice. In addition, PCR analysis was performed on 64 kinds of rice presently available in the Korean market, and no Bt rice was detected. This method presented in this paper can be used as a highly sensitive and specific detection method of Bt rice.