Conventional Escherichia coli (E. coli) detection methods are often time-consuming, while molecular diagnostics typically rely on expensive thermocycling equipment. To address these limitations, this study developed a rapid nucleic acid detection method for E. coli by integrating multienzyme isothermal rapid amplification (MIRA) with Pyrococcus furiosus Argonaute (PfAgo)-mediated targeted cleavage. The conserved housekeeping gene phoA was selected as the target, and specific MIRA primers and 5′-phosphorylated guide DNAs (gDNAs) were designed accordingly. After exponential amplification at 39 °C, the amplicons were specifically recognized by PfAgo at 95 °C, leading to molecular beacon cleavage and generation of a detectable FAM fluorescence signal. Among the tested guides, gDNA6 showed the highest cleavage efficiency. Optimal performance was achieved with 1 μM PfAgo, 0.5 μM gDNA, and 5 mM MnCl₂. The optimized MIRA-PfAgo assay demonstrated a limit of detection of 10⁰ copies/μL, comparable to qPCR, and exhibited high specificity with no cross-reactivity against common enteric pathogens. In 28 clinical and environmental samples, the assay results were fully consistent with those of qPCR. Overall, the MIRA-PfAgo platform provides a rapid, sensitive, and specific approach for E. coli detection, demonstrating strong potential to reduce reliance on precision thermal cyclers for resource-limited applications.