Traditional approaches for the detection and differentiation of Bacillus cereus group species often face challenges due to the complexity of genetic discrimination between species. In this protocol, we propose a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay incorporates a universal fluorescent reporter and four DNA binding fragments, three of which are responsible for “opening up” the folded rRNA while the fourth strand is responsible for detecting single nucleotide variation (SNV) with high selectivity. The binding of the DNM to 16S rRNA results in the formation of the 10-23 DNAzyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. The developed biplex assay enables the detection of B. thuringiensis 16S rRNA and B. mycoides at fluorescein and Cy5 channels, respectively. The protocol offers two detection options: one utilizing extracted total RNA and the other involving crude cell lysate. The latter enables a fast and straightforward detection after 1.5 h with a hands-on time of ~15 min. The new protocol may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis.