CRISPR/Cas-based systems are highly attractive for developing next-generation diagnostic technologies because of their intrinsic merits such as simplicity, sensitivity, and specificity. However, currently, nucleic acid amplification procedures are still needed to achieve attomolar sensitivity in most CRISPR/Cas-based assays, which causes high cost, operation difficulty, and low efficiency. Herein, we combine the CRISPR/Cas12a-based assay and a single-microbead detection platform for one-step and amplification-free detection of DNA at the single-molecule level. By modifying DNA reporters on a biomimetic membrane-coated microbead, the activated Cas12a by targets will cleave these reporters and lighten the bead within 10 min. The method allows the detection of the target down to three copies in a 5 μL sample. Furthermore, we successfully apply this method for the specific identification of viral infection, foodborne bacteria, and DNA mutation in real samples without extra nucleic acid amplification. We believe that this approach offers new insights for developing CRISPR/Cas-based DNA assays in biomedical applications.