Plant miRNAs, the endogenous non-coding small RNAs of about 20-24 nucleotides, play important roles in multiple biological processes by acting as negative regulators of their targeted mRNAs. Soybean (Glycine max (L.) Merr.) is one of the important oil crops of the world, in which many miRNAs have been obtained through the computational prediction or experiments. However, the miRNA genes identified for soybean are still far from saturation, and their biological functions are largely unknown. Here, a total of 48 candidates of miRNAs were identified following a range of strict filtering criteria. Detailed sequence analysis showed that G. max pre-miRNAs vary in length from 47 to 380 nt, embody mature miRNAs that differ in their physical location within the pre-miRNAs. In this study, twenty miRNAs were confirmed by microarray and three miRNAs were further validated by poly(A)-tailed RT-PCR. Comparative sequence analysis of soybean miRNA sequences showed that uracil is the dominant base in the first position at the 5' end of the mature miRNAs, and the base may have an important functional role in miRNA biogenesis and/or miRNA-mediated gene regulation. Finally, we predicted potential targets of these miRNAs. These target genes were predicted to encode transcription factors, resistance protein, heat shock protein, protein kinase, transporter, zinc finger protein and others, which might play important roles in soybean development and stress response.