Tuber dormancy and sprouting are significant for potato cultivation, storage, and processing. Although the substantial role of microRNAs (miRNAs) in some biological processes has been recognized, the critical role of miRNA in breaking potato tuber dormancy is not well understood to date. In this investigation, we expand research on miRNA-mediated gene regulation in tuber dormancy release. In this work, 204 known and 192 novel miRNAs were identified. One hundred thirty-six differentially expressed miRNAs (DE-miRNAs) were also screened out, of which 56 DE-miRNAs were regulated by temperature during tuber dormancy release. Additionally, degradome sequencing revealed that 821 target genes for 202 miRNAs were discovered. Among them, 63 target genes and 48 miRNAs were predicted to be involved in plant hormone signaling pathways. This study used degradome sequencing, tobacco cotransformation system, and β-glucuronidase (GUS) staining technology to confirm that stu-miR319c can target