MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms. The advent of new high-throughput sequencing technologies has enabled the discovery of novel miRNAs. Although there are two recent reports on high-throughput sequencing analysis of small RNA libraries from different organs of two wine grapevine varieties, there was a significant divergence in the number and kinds of miRNAs sequenced in these studies. More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine. In this study, a total of 130 conserved grapevine Vitis vinifera miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, other 80 unconserved Vv-miRNAs including 72 novel potential and 8 known but unconserved ones were found. Fifty-two (52.5%) of these 80 unconserved Vv-miRNAs exhibited differential poly(A)-tailed reverse transcriptase-polymerase chain reaction expression profiles in various grapevine tissues that could further confirm their existence in grapevine, among which 20 were expressed only in grapevine berries, indicating a degree of fruit-specificity. One hundred thirty target genes for 56 unconserved miRNAs could be predicted. The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed. These results point to a regulatory role of miRNAs in grapevine berry development and response to various environments.