Drought stress is considered a critical environmental factor that negatively affects wheat growth and development, which causes considerable losses in wheat yields worldwide. More recently, numerous microRNAs (miRNAs) have been found to be involved in wheat responses to drought stresses. However, there is little information regarding the effects of He-Ne laser irradiation on the expression traits of miRNAs and their targets in wheat seedlings exposed to drought stress. In the current study, therefore, a combination of physiological and molecular approaches was used to assess the effect of He-Ne laser irradiation on the expression of miRNAs and their targets in wheat seedlings exposed to drought stress. Our results demonstrated that drought stress significantly reduced plant height, root length, shoot and root fresh weight, relative water content, the expression level and activity of superoxide dismutase (SOD), enhanced malondialdehyde (MDA) concentration in the wheat seedlings. However, He-Ne laser irradiation significantly enhanced the activities of SOD, ascorbate peroxidase (APX), peroxidase (POD) and relative water content, and reduced MDA concentration of seedlings by regulating gene expression for SOD, POD, APX. In addition, in comparison with drought stress alone, miR160, miR164 and miR398 transcripts were down-regulated, and expression levels of its targets auxin response factor (ARF22), NAC domain transcription factor and Cu/Zn superoxide dismutases (CSD) were up-regulated in He-Ne laser irradiated seedlings exposed to drought stress. These results suggested that He-Ne laser irradiation could possible protection of drought stress, at least partially, by increasing the transcript levels and activities of SOD, POD and APX, and decreasing the transcript levels of miR160, miR164 and miR398. To the best of our knowledge, this is the first study to present biochemical and molecular evidence supporting the effect of He-Ne laser irradiation on the alleviation of drought stress in wheat seedlings mediated by miRNA expression.