Neonicotinoid pesticides have recently drawn attention worldwide owing to their potential adverse effects on non-target organisms and ecosystems. Exposure to imidacloprid, the most widely used neonicotinoid insecticide, is of particular concern among rural populations because of its ubiquitous use in agriculture. Hence, biological monitoring of urinary imidacloprid and its major metabolite 6-chloronicotinic acid (6-CNA) was performed using Polar Enhanced Polymer solid-phase extraction by LC-MS/MS with mean recoveries of 78.3-109.8% and limits of quantitation at 0.029-0.038 ng/mL. Imidacloprid was detected in 100% of urine samples from rural applicators at concentrations of 0.21-8.91 ng/mL (0.06-9.60 μg/g creatinine) and 0.11-24.58 ng/mL (0.66-57.40 μg/g creatinine) before and after pesticide application, respectively. Significant increase in urine concentration (3.52- to 3.77-fold) of imidacloprid and 6-CNA was observed after local imidacloprid field application (p ≤ 0.001). The estimated absorbed daily dose (ADD) for imidacloprid was 0.52-248.05 μg/kg/d, indicating that attention should be paid to potential health risks for applicators because of increased imidacloprid exposure at level of significance (p < 0.05). This study is the first to report ADD estimation for imidacloprid, thereby providing an important reference for further human health risk evaluation.