MicroRNAs (miRNAs) are a class of endogenous noncoding small RNAs that play important roles in various biological processes and diseases. Direct determination of miRNAs is a cost-efficient and accurate method for analysis. Herein, we established a novel method for the analysis of miRNAs based on a narrow constant-inner-diameter mass spectrometry emitter. We utilized the gravity-assisted sleeving etching method to prepare a constant-inner-diameter mass spectrometry emitter with a capillary inner diameter of 5.5 μm, coupled it with a high-voltage power supply and a high-resolution mass spectrometer, and used it for miRNA direct detection. The method showed high sensitivity and reproducibility for the analysis of four miRNAs, with a limit of detection of 100 nmol/L (170 amol) for the Hsa-miR-1290 analysis. Compared with commercial ion sources, our method achieved higher sensitivity for miRNA detection. In addition, we analyzed the total miRNAs in the A549 cells. The result indicated that both spiked and endogenous miRNAs could be quantified with high accuracy. As a result, this method offers a promising platform for highly sensitive and accurate miRNA analysis. Furthermore, this approach can be extended to the analysis of other small oligonucleotides and holds the potential for studying clinical samples and facilitating disease diagnosis.