Blackleg caused by Leptosphaeria biglobosa, is one of the damaging diseases of oilseed rape (Brassica napus). Based on histological observation and RNASeq, we analyzed the phenotypes and gene expression changes of oilseed rape after inoculation with L. biglobosa for 4, 12, 24, 36, 48 and 96 h to discuss the defense response and disease resistance mechanism of oilseed rape in response to the infection of L. biglobosa, provided a theoretical basis for comprehensively revealing the molecular mechanism of interaction between oilseed rape and L. biglobosa, and accumulated genetic information for breeding the disease resistance varieties of oilseed rape. The results showed that: (1) after inoculation for 4-96 h, the leaf spot gradually expanded, and the L. biglobosa formed mycelium within 48-96 h. (2) By RNASeq, a number of 3 384, 2 270, 3 802, 5 811, 6 155, 7 153 differentially expressed genes (DEGs) were obtained at 4, 12, 24, 36, 48 and 96 h of L. biglobosa infection on oilseed rape. (3) qRTPCR results showed that the levels of the 15 differentially expressed genes in oilseed rape were basically consistent with the results of RNASeq. (4) The differentially expressed genes were analyzed by short time series clustering and KEGG enrichment analysis. The results showed that the genes in the pathways of plant and L. biglobosa interaction, protein kinase, jasmonate/ethylene/salicylic acid signaling, and glucosinolate biosynthesis were strongly activated, and their gene expression showed dynamic change trend.