microRNA (miRNA) is a small endogenous noncoding RNA molecule that plays multiple roles in regulating most biological processes. However, for China’s national treasure giant panda, a world-famous rare and protected species, reports of its miRNA have been found only in blood and breast milk. To explore the miRNA expression differences between different giant panda tissues, here, we generated the miRNA profiles of five tissues (heart, liver, spleen, lung, and kidney) from four giant pandas with Illumina Hiseq 2500 platform, and filtered the differentially expressed miRNAs (DEmiRs) in each tissue, predicted the target genes of miRNA from each tissue based on the DEmiRs. Then, the GO and KEGG enrichment analysis were conducted using the target genes predicted from DEmiRs in each tissue. The RNA-seq generated an average of 0.718 GB base per sample. A total of 1,256 known miRNAs and 12 novel miRNAs were identified, and there were 215, 131, 185, 83, and 126 tissue-specific DEmiRs filtered in the heart, liver, spleen, lung, and kidney, respectively, including miR-1b-5p, miR-122-5p, miR-143, miR-126-5p, and miR-10b-5p, respectively. The predicted target genes, including MYL2, LRP5, MIF, CFD, and PEBP1 in the heart, liver, spleen, lung, and kidney, respectively, were closely associated with tissue-specific biological functions. The enrichment analysis results of target genes showed tissue-specific characteristics, such as the significantly enriched GO terms extracellular matrix in the heart and insulin-like growth factor binding in the liver. The miRNA profiles of the heart, liver, spleen, lung, and kidney of giant panda have been reported in this study, it reveals the miRNA expression differences between different tissues of the giant panda, and provides valuable genetic resources for the further related molecular genetic research of the rare and protected species giant panda and other mammals.