The precondition of studying biological sample is to extract sample metabolites by the best pretreatment methods. There is already limited information about pretreatments of fermented feed metabolites. The study compared the extraction effects of different pulverisation methods used in the sample pretreatment process for the extraction of metabolites from cottonseed meal fermented by Lactobacillus acidophilus based on UPLC-Q-TOF-MS. The extraction effects of three pretreatments (non-pulverisation (WF), pulverisation (F), and high-speed homogenisation methods (YJ)) were compared with the numbers of metabolites and the normalised peak areas of the metabolites. The results showed that the number of metabolites extracted with three pulverisation methods were 1745, 1896, 2132 (ESI+ mode) and 1447, 1675, 2073 (ESI- mode), respectively. The number of variable importance plot (VIP) metabolites and the relative peak areas of metabolites showed that the trend was YJ > F > WF. The extraction effect of high-speed homogenisation method was the best way to extract metabolites from the fermented cottonseed meal. This study built a foundation work for the further research of the fermented feed metabolomics.