Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C19-GA2ox and two C20-GA2ox clades. Group 4 is a C20-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.