We constructed small RNA and degradome sequencing libraries to identify miRNAs and targets involved in tomato pedicel abscission, and confirmed their roles via quantitative real-time PCR. MicroRNAs (miRNAs) are endogenous small RNAs which play crucial negatively regulatory roles at both the transcriptional and post-transcriptional levels in plants; however, limited knowledge is available on the expression profiles of miRNAs and their target genes during tomato pedicel abscission. Taking advantage of small RNA (sRNA) and degradome sequencing technology, a total of 56 known and 11 novel candidate miRNAs targeting 223 mRNA genes were confirmed during pedicel abscission. Gene ontology annotation and KEGG pathway analysis showed that these target genes were significantly enriched in intracellular, membrane-bounded organelle-related biological processes as well as in metabolic, plant-pathogen interaction and hormone signaling pathways. We screened 17 miRNA/target pairs for further analysis and performed quantitative real-time PCR to identify the roles. Cluster analysis of selected miRNAs revealed that the expression profiles of miRNAs varied in different stages of abscission and could be impacted by ethylene treatment. In the present study, the correlations between miRNAs and targets suggested a complex regulatory network of miRNA-mediated target interaction during pedicel abscission. Additionally, the expression profiles of miRNAs and their targets changed by ethylene might be a considerable reason why ethylene promotes pedicel abscission. Our study provides new insights into the expression and regulatory profiles of miRNAs during tomato pedicel abscission.