Trimethoprim (TMP), a diaminopyrimidine antibacterial synergist, is widely employed in the prevention and treatment of diseases in livestock and poultry. However, the development of sensitive and rapid detection methods is necessitated by the increasing incidence of TMP residues exceeding regulatory limits. In this study, a time-resolved fluorescence microsphere-based lateral flow immunoassay (TRFMs-LFIA) for the detection of TMP in livestock and poultry products was established. The method was developed based on highly specific monoclonal antibodies against TMP and the unique advantages of time-resolved fluorescent microspheres. Key parameters including microsphere particle size, the pH of the activation buffer, the amount of fluorescent immunoprobe, and the standard diluent were systematically optimized. The TRFMs-LFIA achieved sensitive detection of TMP residues at ppb concentrations within 11 minutes. Under optimal conditions, the limits of detection (LOD) for TMP in milk, egg, and chicken samples were 0.13 ng·mL-1, 0.23 and 0.29 μg·kg-1, respectively ; the linear detection ranges (IC20–IC80) were 0.27~3.10 ng·mL-1, 0.53~2.96 μg·kg-1, 0.37~2.09 μg·kg-1, respectively; and the cut-off value was 12.5 μg·kg-1, with cross-reactivity against structural and functional analogs below 19.48%. Additionally, the average spiked recovery rate for the samples ranged from 82.00% to 125.33%, with coefficients of variation below 15.19%, and was in agreement with the results obtained by HPLC-MS/MS (R2 = 0.96). Collectively, these results indicate that the established TRFMs-LFIA exhibits good accuracy and simple operation, making it suitable for the rapid quantitative detection of TMP in livestock and poultry products and providing reliable technical support for food safety regulation.