Acinetobacter johnsonii is a critical threat to aquatic products. However, the complete genome sequences and metabolome knowledge of A. johnsonii are still limited with respect to spoilage-associated and cold adaptive characteristics. The aim of this study was to report the spoilage and cold adaptive markers of Acinetobacter johnsonii XY27 from spoiled bigeye tuna (Thunnus obesus) by complete genome sequencing and metabolomic and volatile organic compound analysis. Based on genome analysis, A. johnsonii XY27 contained several spoilage-associated genes involved in sulfur metabolism (TauE, SafE, FrdB, TST, iscS), protease secretion (DegP, htrA, clpX, clpP), and biofilm formation (gspE, gspL, gspM, gspD, gspF, gspJ, gspK, RpoN). In addition, several spoilage-related pathways were enriched in glycine, serine and threonine metabolism and histidine metabolism. Growth of A. johnsonii XY27 under cold temperature was characterized by the production of extensive amounts of tryptophan, aminoacetone, 3-methyl pyruvic acid, l-threonine, choline, PS(18:0/18:1(9Z)), and l-serine. Volatile organic compounds such as benzaldehyde, 1-hexanol and 2,4-di-tert-butylphenol might be used as biomarkers of the spoilage potential of A. johnsonii XY27 under cold adaptation. Therefore, the complete genome sequence, metabolomic analysis and gas chromatography–mass spectrometry information provide valuable tools for spoilage mechanism investigations on A. johnsonii XY27.