Gazami crab (Portunus trituberculatus) is prone to spoilage during storage and transportation. More research is needed to determine how to reliably show its freshness and explain the mechanism of quality deterioration. We hypothesized that proteins extracted from crabs can be biomarkers to detect crab muscle quality changes. This work used physicochemical and proteomic approaches to investigate protein biomarkers and molecular mechanisms driving changes in gazami crab muscle quality after long-term refrigeration. It was shown that 66 differentially abundant proteins (DAPs) were closely associated with pH and texture and can be used as biomarkers to assess crab muscle freshness. According to bioinformatics studies, ribosomes and autophagy were significant mechanisms in crab rotting. These findings provided new concepts and a theoretical foundation for evaluating the freshness of refrigerated gazami crab and help uncover the molecular mechanism of its quality deterioration.