A pre-concentrated immunochromatographic assay for Salmonella enteritidis (S. enteritidis) detection was developed based on the unique optical and magnetic properties of magnetic nanoparticles (MNPs). The target was easily concentrated from a large volume of sample by using immune MNPs (IMNPs) with superparamagnetism. The results could be directly distinguished using colored IMNPs on test strip. A target elution step was not required in the proposed method. The key parameters, including coupling pH, blocking agents, and amount of IMNPs on the test strip, were optimized. Colloidal gold-based immunochromatographic assay (CG-ICA), MNP-based immunochromatographic assay (MNP-ICA), and MNP-ICA for a large-volume sample (large-volume MNP-ICA) were compared. Under optimal conditions, the sensitivity of large-volume MNP-ICA was 1.95 × 10⁵ CFU mL⁻¹, which was 10 times higher than that of MNP-ICA and 1000 times higher than that of CG-ICA, owing to the pre-concentration from a large sample volume. Moreover, the antibody consumption of large-volume MNP-ICA was lower than that of conventional CG-ICA. The entire detection time of the proposed method was only 30 min. In general, the proposed method may be considered as a rapid, sensitive, and simple screening tool for on-site detection of S. enteritidis as well as other pathogens.