Salbutamol (SAL) is a short-acting β2-adrenergic receptor agonist (β-agonist) forbidden in livestock production. To develop a more sensitive and specific detection method for SAL, a new hapten was synthesized, keeping the structure of benzene ring side of SAL while changing the tert-butyl group structure that SAL shares with other β-agonists. Ten mice were immunized with a SAL hapten–keyhole limpet hemocyanin (KLH) conjugate, and cells were selected within a urine sample at the cell fusion stage. The best monoclonal antibody (Mab) had an IC₅₀ value of 0.31 ng mL⁻¹ in PBST buffer and an IC₅₀ value of 0.19 ng mL⁻¹ in a swine urine sample diluted five times (with a lower B₀ value), and had no cross-reactivity with clenbuterol and other β-agonists. These results show that the hapten design was successful and that the Mab can potentially be used in highly sensitive immunoassays for specifically monitoring the illegal use of SAL in real samples.