The objectives of the current study were to investigate differences in rumen metabolome and fatty acid (FA) targeted metabolomics in blood and milk in dairy cattle with different sorting behaviors. Twenty-four Holstein cows with an initial average body weight of 581.59 ± 29.7 kg were enrolled in the experiment. Cows were fed and milked three times daily over a 7-day pre-trial period, followed by a 21-day main trial period. Feed intake and feed sorting were measured weekly. Milk yield was recorded on days 7, 14, and 21, and milk samples were collected on days 14 and 21 of the main trial period to analyze milk composition and FA profiles. Blood samples were collected on day 20 to determine FA profiles. Rumen fluid samples were collected on day 21 to measure pH, volatile fatty acids (VFA), and the rumen metabolome. Based on feed sorting behavior, six cows that severely sorted for fine particles (SEV; n = 6) and six cows that slightly sorted for fine particles (SLI; n = 6) were classified into two groups. Comparative analysis revealed differences between the groups, with the SEV group demonstrating lower rumen pH values alongside elevated concentrations of ruminal total VFA and acetate (P < 0.05). Additionally, the SEV group showed a lower dry matter intake (DMI; P < 0.001). In serum FA composition, the SEV group displayed higher proportions of C > 16 and C18:1 cis9 (P < 0.05), while showing markedly lower proportions of C13:0, C14:1 trans9, C14:1 cis9, C16:1 trans9, C16:1 cis9, C18:1 cis11, C22:5 cis7,10,13,16,19, and C24:1 cis15 (P < 0.05). Analysis of milk FA profiles revealed that the SEV group had elevated proportions of C18:1 trans11 and C18:1 trans12, coupled with a reduced C23:0 percentage (P < 0.05). Analysis of milk-to-serum FA ratios indicated that C ≤ 16, C13:0, C14:1 trans 9 + cis 9, and C16:1 trans 9 + cis 9 were higher (P < 0.05), whereas C18:1 cis 9 was lower in SEV cows compared to SLI cows (P = 0.042). The integrated analysis of the ruminal metabolome demonstrated that the SEV group, compared to the SLI group, exhibited elevated concentrations of lactic acid, hypoxanthine, and histamine (P < 0.05), while showing decreased levels of arachidonic acid (P = 0.041). Pathway analysis identified significant alterations (P < 0.05; impact > 0.1) in several key metabolic processes associated with excessive sorting for fine particles, including butanoate metabolism, histidine metabolism, and arachidonic acid metabolism. Generally, these findings provide valuable new insights into the metabolic implications of sorting behavior in dairy cows, enhancing the understanding of the relationship between variation in sorting for fine particles and metabolic processes.