Due to high incidence, poor prognosis, and easy transformation into pancreatic cancer (PC) with high mortality, early diagnosis and prevention of acute pancreatitis (AP) have become significant research focuses. In this work, we proposed a magnetic single-drop microextraction (SDME) system with spatial confinement to enhance the aggregation-induced emission (AIE) effect for simultaneous fluorescence detection of miRNA-155 (associated with AP) and miRNA-196a (associated with PC). The target miRNAs were selectively recognized by the hairpin probe and triggered the DNA amplification reaction; then, the DNA strands with two independent probes of G-quadruplex/TAIN and Cy5 were constructed on the surfaces of the magnetic beads. The SDME process, in which a drop containing the fluorescence probes was formed at the tip of the magnetic microextraction rod rapidly within 10 s, was performed by magnetic extraction. In this way, G-quadruplex/TAIN was enriched owing to the spatial confinement of the single-drop system, and the fluorescence signal given off (by G-quadruplex/TAIN) was highly enhanced (AIE effect). This was detected directly by fluorescence spectrophotometry. The approach achieved low limits of detection of 2.1 aM for miRNA-196a and 8.1 aM for miRNA-155 and wide linear ranges from 10 aM to 10 nM for miRNA-196a and from 25 aM to 10 nM for miRNA-155. This novel method was applied to the fluorescence detection of miRNAs in human serum samples. High relative recoveries from 95.6% to 104.8% were obtained.