MicroRNAs (miRNAs) are endogenous small non-coding RNAs that regulate gene expression at posttranscriptional level. They might regulate tolerance to abiotic stress. However, this has not been studied in Tradescantia (BNL clone 3340), an important bio-indicator for measuring the effect of radiation. Up to date, gamma irradiation (gamma-IR) responsive miRNAs and their expressions patterns in plants in response to gamma-IR stress have not been reported yet. Therefore, putative gamma-IR responsive miRNAs from a cDNA library of Tradescantia after radiation stress were predicted in this study by using comprehensive genomic approaches. Here, we identified 37 miRNAs belonged to 36 different miRNA families. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis of five randomly selected miRNAs were confirmed that their responsiveness to gamma-IR stress. Target prediction revealed that 37 miRNAs targeted 149 genes involved in stress tolerance regulation, light response, redox systems, signaling pathways, DNA repair, and transcription factors. A coordinated reverse expression changes between miRNAs and their target genes further validated that these predicted miRNAs could be gamma-IR responsive and likely to be directly involved in stress regulatory networks of Tradescantia. Altogether, these results will provide novel insights to the understanding of molecular mechanisms of miRNAs involved in plant response to gamma-IR.