Winter rapeseed seedlings are susceptible to low temperature during overwintering in Northwest China, leading to reduced crops production. Freezing stress is one of the main environmental stresses in Northwest China from late autumn to early spring, an eventful period for overwinter survival rate of winter rapeseed. However, the molecular mechanism of freezing tolerance formation is still very backward in winter rapeseed. In this study, using a pair of freezing-sensitive and freezing-resistant cultivars NQF24 and NTS57, the exhaustive effects of freezing stress on freezing tolerance formation were evaluated by analyzing leaf at the levels of transcriptome, proteome, physiology and ultrastructure. There were 8497 and 7358 differentially expressed genes (DEGs) and 418 and 573 differentially abundant proteins (DAPs) identified in the leaf of NQF24 and NTS57 under freezing stress, respectively. Function enrichment analysis showed that most of the enriched DEGs and DAPs were associated with plant hormones signal transduction, fatty acid metabolism, ribosome, plant-pathogen interaction and secondary metabolites biosynthesis. Freezing tolerance is formed by enhanced signals transduction, increased the biosynthesis of protein and secondary metabolites, enhanced reactive oxygen species (ROS) scavenging, more osmolytes, lower lipid peroxidation, and stronger cell stability. These results can be taken as selection indicators in freezing tolerance breeding program in rapeseed.