摘要： Rapid and sensitive foodborne pathogen detection assay, which can be applied in multiple fields, is essential to timely diagnosis. Herein, we proposed a multi-signal readout lateral flow immunoassay for Salmonella typhimurium (S. typhi) detection. The assay employs colorimetric-fluorescent-magnetic nanospheres (CFMNs) as labels, which possess multi-functional: target separation and enrichment, multi-signal readout and two formats of quantitation. The assay for S. typhi detection involves magnetic separation and chromatography. First, the S. typhi were separated and enriched from matrix by antibody labelled CFMNs, and then the S. typhi-containing suspension is added onto the sample pad to flow up the test strip. The introduction of magnetic separation enhances anti-interference ability and 10-fold sensitivity, making the assay possible for practical application. The assay has realized naked eye detection of 1.88×104 CFU/mL S. typhi, and 3.75×103 CFU/mL S. typhi can be detected with a magnetic assay reader, which is 2–4 orders of magnitude lower than other label-based LFIAs, with a quantitation range of 1.88×104 to 1.88×107 CFU/mL by measuring the fluorescence intensity and magnetic signal. Moreover, the successful detection of S. typhi in complex matrix (tap water, milk, fetal bovine serum and whole blood) indicated its potential application in real samples.