<p>Nowadays, specific biomarkers and detection methods for the early, rapid and non-invasive diagnosis of Alzheimer’s disease (AD) are scarce. MiR-34a and miR-206 are both upregulated in the cerebrospinal fluid and peripheral fluids of AD patients. In view of the similar biochemical information between interstitial fluid (ISF) and blood and high proportions of RNAs in ISF, this work developed an integrated optical patch that combined hydrogel microneedles (MNs) extraction with on-site fluorescence detection for the simultaneous sensing of miR-34a and miR-206. A zwitterionic compound trimethylamine nitrogen oxide amphiphile (TMAO) was introduced to the matrix of MNs by co-modification with hyaluronic acid (HA) and methacrylic acid (MAA) to endow the novel MNs with adjustable swelling ability, as well as resistance to non-specific adsorption property. Moreover, to avoid signal interferences, we innovatively performed spatial average cutting on the MNs. By embedding two toehold-mediated strand displacement (TMSD) probes within MNs matrix, a dual-emission MNs optical patch for the two miRNAs was thereby constructed. As a potential non-invasive and accurate diagnostic tool of AD, the capability of the proposed optical patch was further demonstrated by simultaneously and on-site differentiating expression levels of miR-34a and miR-206 in ISF between normal and AD model mice.</p> Graphical Abstract <p></p>