Near infrared spectroscopy (NIRS) has been proved to be a powerful analytical tool in different fields. However, because of the low sensitivity in near infrared region, it is a significant challenge to detect trace analytes with normal NIRS technique. A novel enrichment technique called fluidized bed enrichment has been developed recently to improve sensitivity of NIRS which allows a large volume solution to pass through within a short time. In this paper, fluidized bed enrichment method was applied in the determination of trace dimethyl fumarate in milk. Macroporous styrene resin HZ-816 was used as adsorbent material, and 1 L solution of dimethyl fumarate was run to pass through the material for concentration. The milk sample was pretreated to remove interference matters such as protein, fat, and then passed through the material for enrichment; after that, diffuse reflection NIR spectra were measured for the analyte concentrated on the material directly without any elution process. The enrichment and spectral measurement procedures were easy to operate. NIR spectra in 900-1,700 nm were collected for dimethyl fumarate solutions in the concentration range of 0.506-5.060 μg/mL and then used for multivariate calibration with partial least squares (PLS) regression. Spectral pretreatment methods such as multiplicative scatter correction, first derivative, second derivative, and their combinations were carried out to select the optimal PLS model. Root mean square error of cross-validation calculated by leave-one-out cross-validation is 0.430 μg/mL with ten PLS factors. Ten samples in an independent test set were predicted by the model with the mean relative error of 5.33%. From the results shown in this work, it can be concluded that the NIR technique coupled with on-line enrichment method can be expanded for the determination of trace analytes, and its applications in real liquid samples like milk and juice may also be feasible.