Brassica napus L. (B. napus) is a major edible oil crop grown around the southern part of China, which often faces cold stress, posing potential damage to vegetative tissues. To sustain growth and reproduction, a detailed understanding of fundamental regulatory processes in B. napus against long-term low temperature (LT) stress is necessary for breeders to adjust the level of LT adaption in a given region and is therefore of great economic importance. Till now, studies on microRNAs (miRNAs) in coping with LT adaption in B. napus are limited. Here, we performed an in-depth analysis on two B. napus varieties with distinct adaptability to LT stress. Through integration of RNA sequencing (RNA-seq) and small RNA-sequencing (sRNA-seq), we identified 106 modules comprising differentially expressed miRNAs and corresponding potential targets based on strong negative correlations between their dynamic expression patterns. Specifically, we demonstrated that Bna-miR397a post-transcriptionally regulates a LACCASE (LAC) gene, BnaLAC2, to enhance the adaption to LT stresses in B. napus by reducing the total lignin remodelling and ROS homeostasis. In addition, the miR397-LAC2 module was also proved to improve freezing tolerance of Arabidopsis, indicating a conserved role of miR397-LAC2 in Cruciferae plants. Overall, this work provides the first description of a miRNA-mediated-module signature for LT adaption and highlights the prominent role of laccase in future breeding programme of LT tolerant B. napus.