Currently, aquatic products are mainly transported through cold chain logistics, which has been proven to significantly reduce the deterioration of quality and freshness. This study aimed to investigate the mechanisms of quality deterioration in large yellow croaker fillets under different cold chain conditions, specifically focusing on temperature fluctuations. Quality changes in the fillets were assessed through microbiological indicators and physicochemical properties. High-throughput sequencing was used to identify the microbial community structure, and LC-MS-based untargeted metabolomics identified the distinctive metabolites in the fillets under cold chain conditions. Notably, the group experiencing the greatest temperature fluctuations exhibited the highest pH value (7.15), trichloroacetic acid (TCA)-soluble peptides content (10.57 μmol tyrosine/g), total volatile basic nitrogen (TVB-N) content (34.91 mg N/100 g), and hypoxanthine (Hx) content (4.70 μmol/g), along with the lowest hypoxanthine ribonucleoside (HxR) content (1.67 μmol/g) and water holding capacity (WHC) value (79.22%). High-throughput sequencing results showed that the relative abundance of Aeromonas reached 54.01-56.95% in the temperature fluctuation groups on day 7.5. Additionally, the sharpest decrease in the relative abundance of Shewanella in temperature fluctuations groups was 70.16% lower than in the constant temperature group. Bioinformatics analyses further confirmed that amino acid metabolism was the most affected metabolic pathway due to temperature fluctuations in cold chain logistics. This study provides new insights that can improve the practical implementation of cold chain storage and preservation of fish.