Seafood substitutions is a global problem and come under the spotlight in recent years. In Taiwan, Greenland halibut is usually substituted for the cod because of its lower price. Nowadays, DNA technology is widely used for fish species identifications; however, it still has concern about the DNA of processed fishery products might be destroyed. This study was designed to develop a proteomic-based method for fish and fishery product authentication by using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) with Sequential window acquisition of all theoretical fragment ion spectra (SWATH). The protein biomarkers from the meat of Alaska pollock, Atlantic cod, and Greenland halibut were identified and validated for species authentication of cod and corresponding fishery products, which might prevent consumer substitutions and fish product mislabeling. Besides, the E. coli proteins can be measured from existing SWATH-MS data though retrospective analysis successfully, it might present the quality of fish meat.