The objective of this paper was to investigate the metabolites of fermented protein feed with different microbial cottonseed meal mixed substrates by the metabolomic analysis platform based on liquid chromatography-mass spectrometry (LC-MS). The experiment used the LC-MS platform to analysis the metabolites which collected from control group, Candida tropicalis group, Saccharomyces cerevisiae group and compound fermented group, and the partial least square-discriminant analysis (PLS-DA) was used to pattern recognition and found small molecular difference metabolites. The results showed that the metabolite relative contents had significant differences in experimental groups compared with the control group, and the metabolites were separated well by PLS-DA. It mainly displayed in carbohydrate metabolites such as mannitol, succinic acid, etc; lipid metabolites such as phosphocholine, L-carnitine, glycerylphosphate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), etc; protein and amino acid metabolites such as dipeptide, tripeptide, betaine aldehyde, homocysteine, etc; and other pathway metabolites such as niacin, ferulic acid, uracil, glycolaldehyde, etc. The relative contents of these metabolites in experimental groups were all significantly higher than those in control group (P<0.05), but the relative contents of glycerylphosphate, PE and PC in experimental groups were significantly lower that than those in control group (P<0.05). In conclusion, a large number of small molecular metabolites, which are produced by different metabolic pathways such as carbohydrate, lipid, protein and others material, are contained in feeds fermented via various strains, and those contents vary with different strains.