Shiga toxin-producing Escherichia coli (STEC) in the environment has been reported frequently. However, robust detection of STEC in environmental samples remains difficult because the numbers of bacteria in samples are often below the detection threshold of the method. We developed a novel and sensitive immuno-PCR (IPCR) assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants. The assay involves immunocapture of Stx2 at the B subunit and real-time PCR amplification of a DNA marker linked to a detection antibody recognizing the Stx2 A subunit. The qualitative detection limit of the assay is 0.1 pg/ml in phosphate-buffered saline (PBS), with a quantification range of 10 to 100,000 pg/ml. The IPCR method was 10,000-fold more sensitive than an analogue conventional enzyme-linked immunosorbent assay (ELISA) in PBS. Although the sensitivity of the IPCR for detection of Stx2 was affected by environmental sample matrices of feces, feral swine colons, soil, and water from watersheds, application of the IPCR assay to 23 enriched cultures of fecal, feral swine colon, soil, and watershed samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. The modification of the sandwich IPCR we have described in this study will be a sensitive and specific screening method for evaluating the occurrence of STEC in the environment.