<p>MicroRNA (miRNA) has emerged as a promising class of non-invasive and sensitive biomarkers for cancer diagnosis due to its close association with tumorigenesis. However, the accurate detection of miRNA remains challenging owing to its short sequence, high sequence homology among family members, and low abundance in biological samples. To address the demands of early cancer diagnosis and clinical prognosis monitoring, this study designed and fabricated a colorimetric microfluidic paper-based analytical device (µPAD) for the quantitative detection of miRNA-21. Leveraging the inherent porous structure and ease of functionalization of paper-based materials, along with the specific binding of streptavidin and biotin, this chip integrates microfluidic technology with the peroxidase-mimicking activity of gold-platinum core-shell nanoparticles (Au@Pt NPs) to achieve rapid and sensitive detection of miRNA-21. In the presence of the target miRNA-21, it preferentially hybridizes with the complementary DNA (cDNA), competitively releasing the pre-hybridized Au@Pt NPs-cDNA conjugate. This conjugate then migrates via capillary action to the detection zone, where the Au@Pt NPs catalyze the oxidation of the colorless 3,3’,5,5’-tetramethylbenzidine, producing a deep blue product, thereby converting the biological recognition event into a detectable optical signal. The miRNA-21 concentration can be conveniently quantified by capturing the image with a smartphone and analyzing the grayscale intensity using the freely available Image J software. Under optimized conditions, the proposed µPAD demonstrated a linear detection range from 1 to 2000 nmol/L with a limit of detection of 0.25 nmol/L. The method also showed good specificity and was successfully applied to the detection of miRNA-21 spiked in human serum samples. This work presents a rapid, simple, and cost-effective paper-based platform with considerable potential for point-of-care testing in early cancer diagnosis.</p>