Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect ≥0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1× and 10× diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.