Alfalfa, an important legume forage, is an ideal crop for sustainable agriculture and a potential bioenergy plant. Drought, one of the most common environmental stresses, substantially affects plants’ growth, development and productivity. MicroRNAs (miRNAs) are newly discovered gene expression regulators that have been linked to several plant stress responses. To elucidate the role of miRNAs in drought stress regulation of alfalfa, a high-throughput sequencing approach was used to analyze 12 small RNA libraries comprising of 4 samples, each with 3 biological replicates. We identified 348 known miRNAs, belonging to 80 miRNA families, from the 12 libraries and 281 novel miRNAs using Mireap software. 18 known miRNAs in roots and 12 known miRNAs in leaves were screened out as drought-responsive miRNAs. Except for miR319d and miR157a which were upregulated under drought stress, the expression pattern of drought-responsive miRNAs were different between roots and leaves in alfalfa. This is the first study discovering miR157a, miR1507, miR3512, miR3630, miR5213, miR5294, miR5368 and miR6173 are drought-responsive miRNAs. Target transcripts of drought-responsive miRNAs were computationally predicted. All 447 target genes for the known miRNAs were predicted using an online tool. This study provides a significant insight on understanding drought-responsive mechanisms of alfalfa.