Abstract: To optimize the culture conditions and fermentation parameters of Cetobacterium somerae NK01 (sp. nov.), and to analyze the metabolic profile of fermented soybean meal, the present study conducted systematic research using this strain. The optimal culture conditions for NK01 were determined by single-factor and response surface tests as follows: temperature 40℃, pH 8.0, sucrose as carbon source, yeast extract as nitrogen source, and inoculation volume of 2.7% (v/v). Fermentation trails using different substrates showed that soybean protein and mixed substrates yielded the most optimal fermentation results. Compared with the control, the soybean protein group exhibited a 4.92% increase in crude protein, a 1.64% increase in amino acid content, and a 31.36% decrease in starch content after fermentation with NK01. The mixed-substrates group exhibited a 10.41% increase in crude protein and an 8.37% increase in amino acid content, along with a 23.14% decrease in starch content. Cetobacterium fermentation did not significantly affect the Bacillus counts in the mixed substrate (P>0.05), with the fermented group showing a slight reduction compared to the control. No mold was detected in any sample. Enzyme activity assays revealed that protease, amylase, and phospholipase activities were significantly higher in the soy protein, corn protein, and mixed-substrate fermentation groups than those in the other substrate fermentation groups (P<0.05). NK01 fermentation significantly raised the crude protein levels across all five feed formulas compared with the control, with an optimal fermentation moisture content ranging from 55% to 65%. Response surface optimization showed that temperature was the most significant factor affecting feed fermentation by strain NK01, followed by moisture content. The model-predicted optimal conditions for fermentation of formula 3 were 40℃, 55% moisture content, and an inoculation volume of 30%. Non-targeted metabolomics analysis indicated that NK01 fermentation significantly increased the levels of several beneficial metabolites in soybean meal, including carnosine, succinic acid, phenylacetic acid, and γ-aminobutyric acid. These findings highlight the potential of NK01 in improving feed nutritional quality and promoting host health. Overall, this study provides theoretical and technical support for the large-scale application of Cetobacterium in aquaculture fermented feed production.