In this study, a magnetic relaxation switching (MRS) biosensor was developed for one-step detection of pathogenic bacteria Vibrio parahaemolyticus (VP). The biosensor is based on a click chemistry-mediated sol-gel system and utilizes low-field nuclear magnetic resonance (LF NMR) detection. VP aptamer (Apt)-coated hollow mesoporous silica microspheres (HMSMs) with saturated sodium ascorbate (SAA) solution sealed inside, HMSMs@SAA&Apt, are designed as the signal unit. When the target VP is present, the Apt binds to VP and releases SAA from the signal unit. The released SAA reduces Cu2+ in the test solution to Cu+, which catalyzes the click chemistry reaction between azide-modified polyethylene glycol (PEG-Azide) and acetylene-modified polyacrylic acid (PAA-Alkyne). The sol-gel transition is triggered, a large amount of "free" water is converted into "bound" water, resulting in the decrease of transverse relaxation time (T2). Under the optimal experimental conditions, the variation of T2 (ΔT2) was linearly related to the logarithm of VP concentration in the range of 10 ∼ 1.0 × 108 CFU/mL, with a limit of detection of 5 CFU/mL. The assay demonstrated good selectivity, stability, and reproducibility. It is completed in one step, holding promising applications in the rapid field detection of pathogenic bacteria.