Herbal tea is a highly popular and widely consumed beverage. However, a pain-relieving and fever-reducing drug, acetophenetidin, was recently found to illegally occur in herbal tea for a fraud purpose. Due to the potential health risk and urgent requirement for on-site screening method, a one-step and high specificity strip for identifying acetophenetidin was developed for the first time. Assisted by computational chemistry, four haptens were designed to prepare immunogens and coating antigens for antibody generation, and a specific antibody with ultra-sensitivity and high specificity was generated, showing half maximal inhibitory (IC) of 16.46 ng/mL for acetophenetidin, less than 3.5% of cross-reactivity to analogs by ELISA. A gold nanoparticles immunochromatographic strip was developed for detection of acetophenetidin in herbal tea, demonstrating a cut-off value of 160 ng/mL and a limit of detection of 1.63 ng/mL. The recovery rates were ranged from 102.2% to 106.1% with coefficient of variation between 2.21% and 7.20%. The analysis of real samples (n = 20) by the strip was well correlated with that of the confirmatory method, liquid chromatography−tandem mass spectrometry. The proposed strip has the potential to be used for rapid screening of acetophenetidin in herbal tea.