In recent years, the diseases of Carassius auratus gibelio have occurred frequently, especially with the decrease of water temperature in winter and the decline of fish immunity, which made the pathogenic bacteria easy to invade the fish and led to the outbreak of fish disease and the decline of culture efficiency. To determine the cause of the sudden death of C. auratus gibelio in Jiangsu during the overwintering period, high-throughput sequencing was used to compare the diversity and structure of the microbial community between healthy samples and diseased samples. The types and characteristics of bacteria in the outbreak of disease were analyzed. The results showed that the abundance of Flavobacterium was the highest in the diseased samples at the genus level. At the species level, the abundance of F. psychrophilum in the diseased samples increased significantly, reaching 63.01% and 61.31% respectively, which was significantly higher than that in the healthy group (1.55%). According to the results of the analysis of microbial community characteristics, the dominant pathogen NJ01 was isolated from the lesions on the body surface of the diseased samples. Through bacterial morphology, physiological and biochemical analysis, and 16S rDNA sequence alignment, the NJ01 strain was identified as F. psychrophilum. After 14 days of artificial infection with the NJ01 strain, the mortality rate of the 1.7×10⁶ and 1.7×10⁷ CFU/mL groups reached 100%, and the symptoms of infection were consistent with those of natural disease. Histopathological observation showed that the muscle cells of the diseased fish were necrotic and the stroma was full of lymphocytes; the large cells in the liver were lytic and necrotic, and the nucleus melted; splenocytes were found with scattered necrosis in the spleen with symptoms of hyperemia; the renal tubular epithelium fell off; there were a lot of lymphocytes in the renal interstitium. The results of the drug sensitivity test showed that the NJ01 strain was sensitive to cefoxitin, cefoperazone, gentamicin, and clarithromycin. The conclusion was that the strain NJ01 was the pathogen for the death of C. auratus gibelio, which could disturb the normal immune and metabolic function, and consequently the pathological phenomena of tissues and organs or death appeared. In this study, the pathogenicity of F. psychrophilum in C. auratus gibelio is reported for the first time, which will provide a reference basis for the study of drug prevention and the pathogenesis of “overwintering mortality syndrome" in conventional freshwater fish.