DNA has proven to be exquisite molecules for the design of artificial analytical tools in molecular diagnosing of human diseases. In this work, we constructed a time-resolved strand displacement amplification (TR-SDA) for molecularly determination of microRNA-146a (miR-146a), a potential biomarker of Type 2 diabetes mellitus (T2DM). Such an amplification strategy makes use of a multi-functionalized cytosine-rich hairpin probe (C-HP). Unlike conventional amplifications that relying on exogenous primer-directed strand polymerization, the hybridization of target miR-146a with C-HP causes a C-HP based intramolecular replication in parallel with an miR-146a/C-HP based intermolecular replication. This feature leads to a time-resolved amplification pattern responsible for controlling C-HP with a cascaded signal amplification ability. Without requirement of any assist probes or complex sequence arrangements, the method enables efficiently accumulate numbers of G-quadruplexes at a constant temperature. After intercalated with thioflavin T (ThT), the complex of G-quadruplex/ThT immediately becomes fluorescence-emissive for detecting of the miR-146a concentration. We demonstrated the successful detection of miR-146a with a high sensitivity and also measured miR-146a from healthy donors and T2DM patients with different expression level. As a proof-of-concept study, the TR-SDA acting as a powerful molecular diagnostic method might provide promising opportunities to meet future clinical needs in biomedicine.