Alpha/beta hydrolase (ABHs) fold esterase/lipase proteins represent a prominent family within the serine hydrolase (SH) superfamily that includes esterases and lipases and other catalytic and non-catalytic proteins. ABHs play crucial roles in both the fundamental and secondary metabolic pathways, including the synthesis and degradation of triacylglycerols (TAGs), key components of plant oils. Despite their importance in oil production, the ABH gene family in the oil crop Brassica napus has not been comprehensively analyzed. In the present study, we identified 777 BnABH genes in the B. napus cultivar ‘Zhongshuang 11’ (ZS11). Phylogenetic analysis categorized these BnABH genes into 10 distinct groups. Twenty-four BnABHs were identified through esterase activity staining and mass spectrometry, 11 of which were classified into clade C3. Examination of the gene and protein structures, expression patterns, and cis-elements of the BnABHs in clade C3 suggested diverse functional roles across different tissues and in response to various environmental stresses. In particular, BnABH205 was highly induced by high temperatures. Subcellular localization analysis revealed that the BnABH205 protein was localized to the plastid. Further analysis revealed five haplotypes within the coding and 3′ untranslated regions of BnABH205 that were significantly associated with seed oil content (SOC). Overall, this study provides a comprehensive understanding of BnABHs and introduces a robust methodology for identifying potential esterase/lipase genes that regulate seed oil content (SOC) in response to environmental hazards, especially heat waves during seed maturation.