Vibrio fluvialis is an emerging foodborne pathogen associated with severe infections. In this study, immunomagnetic beads (IMBs) were synthesized by conjugating nanobody N71 to magnetic nanoparticles (MNPs) via polyethylenimine (PEI) and glutaraldehyde (GA) cross-linking. Due to the high affinity of N71 for V. fluvialis lipopolysaccharide, the IMBs efficiently and specifically captured the target pathogen. When integrated with ToxR-targeted quantitative real-time PCR, this system achieved enhanced detection sensitivity (48 CFU/mL) and reduced false-positive rates. The optimization results showed that the capture efficiency of V. fluvailis reached its maximum (95%) when 125 µg of N71 were conjugated to 1 mg of MNPs to form the MNPs-PEI-GA-Nbs conjugates, achieved with a conjugate dosage of 0.5 mg and an incubation time of 45 min. Nontarget bacterial interference experiments and actual sample detections validated the excellent specificity of the method for detecting V. fluvialis. Compared with conventional culture-based methods, this method reduced detection time from 24–48 h to <7 h, providing an efficient and reliable alternative for rapid detection of V. fluvialis in food samples.
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