The freshness of large yellow croaker ( Pseudosciaena crocea ) fillets was evaluated over 20 days of chilled storage under vacuum packaging. The physicochemical properties [pH, K value, thiobarbituric acid (TBA), texture profile analyses (TPA), color], microbiological properties [total viable count (TVC)], sensory attributes, transcriptomics (levels of transcripts coding for cathepsin L), and functional properties of proteins [emulsion activity (EA) and emulsion stability (ES)] were assessed at 0, 5, 10, 15, and 20 days of storage. This study also investigated the effect of the storage time on fish muscle proteome in large yellow croaker using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). The parameters that were the most sensitive to storage duration were color (C*(ab)), TPA (springiness, chewiness, and resilience), pH, K value, TVC, levels of transcripts coding for cathepsin L, EA, ES, and sensory attributes. The three altered proteins were successfully identified. Therefore, these parameters might be considered suitable indicators for evaluating the freshness of large yellow croaker fillets during chilled storage under vacuum packaging.