<p>The expression levels of miRNAs in cells or biological fluids are closely associated with the onset and progression of many diseases, making the detection of miRNAs in serum and plasma increasingly important. However, due to the low levels of miRNAs in biological fluids and the complexity of their surrounding environment, their analysis faces challenges related to accuracy and reproducibility. We developed a duplex-specific nuclease (DSN)-mediated detection method for the direct detection of miRNA-21 in real samples. This method eliminates the need for total RNA extraction from real samples using commercial kits, a key advantage over traditional methods requiring pre-treatment. As verified by performance evaluation, this method exhibits a linear range of 5.00 fmol·L^− 1 to 500 pmol·L^− 1 and a limit of detection (LOD) of 2.66 fmol·L^− 1 for miRNA-21. Direct detection of miRNA-21 in real samples yielded favorable results. These findings confirm that the method holds great application potential for the direct detection of miRNA-21 in real biological samples and exhibits promising prospects in clinical cancer diagnosis and drug screening.</p>