Proline is an important osmotic adjusting material greatly accumulated under drought stress and can help plant to adapt to osmotic stress. MicroRNAs (miRNAs) are small, endogenous RNAs that play important regulatory roles in plant development and stress response by negatively affecting gene expression at post-transcriptional level. Three genes of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR) and proline dehydrogenase (ProDH) are regulating proline metabolism. Until now, little is known about miRNAs regulating proline accumulation. In this work, in order to understand whether miRNAs related to mRNAs of enzymes to regulate proline enrichment under drought stress, we used mRNAs of related enzymes as the targets of miRNAs to search miRBase using BLAST and find many query miRNA sequences. After a range of filtering criteria, 11 known miRNAs classified into 6 miRNA families were predicted. The result from qRT-PCR assay showed that 10 out of 11 predicted miRNAs were successfully detected including 9 down-regulated miRNAs and one up-regulated miRNA. Based on expression and functional analysis, we identified miR172, miR396a, miR396c and miR4233 may regulate P5CS gene, and miR2673 and miR6461 may regulate P5CR and ProDH gene, respectively. The findings can help us make a good understanding of the roles of miRNAs in regulation of proline accumulation and provide molecular evidence for involvement process of drought tolerance in potato.