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Screening for ″zero tolerance″ β-agonists requires broad-specificity and sensitivity methods. Herein, R-(−)-salbutamol (SAL) is chirally separated and designed as a hapten, and a monoclonal antibody (mAb) was first prepared with an IC50 of 0.27 ng/mL, which can recognize 38 β-agonists simultaneously. The broad-specificity of chiral mAb was explored by molecular simulation technology. Magnetic nanoparticles (MNPs) were then synthesized and applied as a signal tracer to develop a lateral flow immunoassay (LFIA). The limits of detection of MNPs-LFIA for SAL in swine urine and pork were 0.05 and 0.09 μg/kg, which was (2–125)-fold lower than that of the reported LFIAs. The recoveries were between 95.8 and 116.7%, with the coefficient of variation from 2.7 to 15.4%. Parallel analysis of 44 samples by commercial ELISA kits confirmed the reliability. Therefore, our work not only provides a broad-specificity and ultrasensitive method for β-agonists but also suggests that chirality is the new general theory that guided the rational hapten design.