Immunomagnetic nanobeads (IMNBs) have been widely used to isolate and detect microorganisms. In this study, recombinantly expressed outer membrane proteins LI0902 and LI1024 of Lawsonia intracellularis (L. intracellularis) were used to yield polyclonal antibodies. IMNBs functionalized with anti-LI0902 polyclonal antibodies display the capture activity to L. intracellularis that was indicated by immunofluorescence assay based on anti-LI1024 polyclonal antibody. One-step quantitative PCR (qPCR) target of the aspartate ammonia-lyase gene based on IMNBs was developed and evaluated using several pathogens associated with pig diarrheal diseases and clinical fecal samples. The results showed that IMNBs had good specificity and sensitivity, no cross-reactivity with other pathogens, and the detection limit was as low as 3.6×10² copies/mL. Meanwhile, IMNBs showed good capture efficiency, 4.21×10⁵ copies of L. intracellularis could be captured by 1µg of IMNBs. Moreover, L. intracellularis in fecal samples were successfully detected by the qPCR combined with IMNBs. The results demonstrate that this method is promising in rapidly and effectively detecting L. intracellularis in clinical fecal samples.