MicroRNAs (miRNAs) are RNA molecules consisting of 20–24 nucleotides that play important roles in regulating plant's gene expression for growth and development, cell viability and stress responses. Viral infection often has a noticeable influence on host gene expression, which may result in a range of developmental abnormalities. To investigate the molecular mechanisms underlying viral infection, miRNA pathway and host gene expression, we report herein the application of the novel miRNAs quantification method in tomato, using a stem-loop reverse transcription followed by SYBR Green PCR assay. For the seven tested miRNAs of Solanum lycopersicum, which are related to the regulation of plant development, hormone response, and their own biogenesis, this quantification method showed high sensitivity, specificity, and wide dynamic range. Precise quantification could be achieved with as little as 0.01 ng of total RNAs for most cases. Additionally, their target mRNAs could be quantified from the same RNA sample simultaneously, by the conventional real-time RT-PCR assay. In comparison with mock inoculation, accumulation levels of the tested miRNAs and target mRNAs were found obviously altered in tomato seedlings, indicating that the miRNA pathway was interrupted by Cucumber mosaic virus and Tomato aspermy virus infection.