MicroRNAs are ~22 nt non-coding endogenous RNAs which play important regulation roles in various species. By using homology-based computational research, 14 novel and conserved tomato miRNAs belonging to ten families were identified from EST, GSS, and nucleotide sequences. Real-time PCR analysis of these miRNAs demonstrated their expression in tomato afterwards. Meanwhile, a total of 36 potential targets were predicted for the ten miRNAs using psRNATarget. The target genes were mainly involved in metabolism, transmembrane transport, stress response, and transcription regulation. According to our experiment, miR398 was down-regulated on different levels under biotic and abiotic stresses, suggesting that miR398 might be involved in tomato stress regulatory network. Our results supplement the findings of tomato miRNAs and also suggest crucial regulatory functions of miRNAs in stress responses.