In　this　work,　high-speed　capillary　sieving　electrophoresis　with　laser-induced　fluorescence　detection　was　applied　to　simultaneously　determine　three　microRNAs.　A　developed　manual　sample　introduction　device　for　the　high-speed　capillary　electrophoresis　system　was　applied　to　perform　sample　injection.　Strategies,　including　field-amplified　sample　injection　and　electrokinetic　injection,　were　studied　to　improve　the　detection　sensitivity.　Under　the　optimal　conditions,　the　limit　of　detection　for　DNA-159　could　be　lowered　to　5.10 × 10−12 mol/L.　In　order　to　achieve　enough　separation　resolution,　two　DNA　probes　were　designed　to　have　extra　sequences　that　acted　as　the　drag　tails.　Under　the　optimized　conditions,　the　three　DNA　probes　and　the　complexes　of　microRNA-156,　microRNA-159,　and　microRNA-166　could　be　completely　separated　within　3.2 min　in　background electrolyte　(pH　8.7)　containing　2.0%　m/m　polyvinyl　pyrrolidone　and　0.4% m/m　hydroxyethyl　cellulose.　The　limits　of　detection　for　the　three　microRNAs　were　0.051,　0.11,　and　0.25 nmol/L,　respectively.　Then　the　method　was　applied　to　analyze　the　microRNAs　spiked　in　the　samples　extracted　from　banana　leaves.　The　recoveries　ranged　from　114.3　to　121.1%　(n = 3).　The　results　showed　that　the　method　developed　in　this　work　was　an　effective　means　for　microRNA　assay.　©　2018　WILEY-VCH　Verlag　GmbH　&　Co.　KGaA,　Weinheim