In order to understand the sequence characteristics of miRNA precursors (premiRNAs) and their expression in different tissues and pseudobulbs infected by soft rot in Oncidium hybridum, we analyzed 25 premiRNAs and 15 mature sequences in the transcriptome and miRNA database of Oncidium. And the expression of 25 premiRNAs in different tissues and pseudobulbs infected by soft rot pathogen were analyzed by realtime fluorescence quantitative PCR (qRTPCR). The results showed that：（1） 25 premiRNAs sequences of Oncidium could be divided into 13 families, including 15 mature sequences.（2）Mfold prediction results showed that all of the Oncidium 25 premiRNAs could form a typical stable stem loop structures with a minimal folding free energies (dG) from -32.04 to -120.98 kal/mol. （3） Sequence alignment analysis showed that there was a conserved region of about 20 bases in the same family of precursors and mature sequences, suggesting that this region may be mature miRNAs. Among them, miRNAs from 10 of 13 precursor families can be fully localized in their precursors.（4） qRTPCR results showed that premiRNAs such as miR159unigene0037857, miR167unigene0011236, miR167unigene0002619, miR169unigene0022341, miR172unigene006514, miR396unigene19032, miR398unigene0009996, miR2950unigene0006151 and miR2950unigene0006422 accumulated in leaf may be helpful for the morphogenesis of the leaves. miR162unigene0003615, miR162unigene0013441, miR166unigene 0011870 and miR168unigene0009958 were highly expressed in roots and leaves, which may be beneficial to the development of roots and leaves. Meanwhile, the abundance expression of miR171unigene 0045985, miR396unigene0011179 and miR396unigene0011180 in roots and pseudobulbs may be beneficial to the development of roots and pseudobulbs.（5） In pseudobulbs infected by soft rot pathogen, the expression levels of miR159unigene0037857, miR167unigene0011236, miR396unigene0011179, miR396unigene0011180, miR396unigene0019032 and miR845unigene0012489 showed atrend of decrease and reached the lowest level after 12 h infection. The expression of miR162unigene0040566, miR166unigene0011870, miR168unigene0009958 and miR171unigene 0045985 increased in pseudobulbs infected by soft rot pathogen after 4 h, and then downregulated. The expression of miR162unigene 0003615, miR167 unigene0002619, miR168 unigene0047942, miR169unigene0022341 and miR845unigene0012489 showed the tendency of first decrease and then increase in the process of pathogen infection, and reached the minimum after 8 h. The above results showed that different members of the 13 families of the premiRNAs of Oncidium are highly conserved and specific in their evolutionary processes, and may be widely involved in the growth and development of different tissues and in response to soft rot pathogen infection in Oncidium.