Sclerotinia sclerotiorum is one of the most important plant pathogens, causing enormous losses in a variety of economically important crops including Brassica napus. The interaction of S. sclerotiorum with its hosts is more complex than initially thought, and still poorly understood. In this study, transcriptome analysis was conducted using S. sclerotiorum RNA from the leaf of B. napus. We mapped 11,074,508 and 11,495,788 paired-end reads. A total of 13,313 genes were obtained and classified according to their functional categories. At 6 h post inoculation (hpi) and 24 hpi, the majority of the upregulated differentially expressed genes (DEGs) were focused on DNA binding and ATP binding. However, the genes under the category of ion binding and oxidoreductase activity were also activated at 24 hpi. Most of the upregulated DEGs at 48 hpi were classified in the category of hydrolase activity. The expression levels of these genes related to hydrolase function were analyzed. The results showed that different genes were activated at different stages. The relative expression level of parts of interesting genes which were activated during the infection process was evaluated by quantitative real-time PCR (qRT-PCR). Our results provide new insight into the infection mechanism of S. sclerotiorum.