The adsorption type and mode of biochar have been extensive research. However, as an indispensable link to reveal the adsorption mechanism, the study of the adsorption sequence is greatly lacking, especially for different pollutants, the way of adsorption is different. In this study, the observation of the adsorption sequence was amplified by KOH modification and optimization of experimental conditions. Persistent radicals (PFRs) combined with liquid mass spectrometry (LC-MS) eliminated interference from oxidation-reduction. Notability, two-dimensional correlation spectroscopy (2D-COS), Raman and Fourier-transform infrared (FTIR) spectroscopy reveals that the vibration amplitude and velocity of the hydroxyl group are better than those of the carboxyl group, methyl group, and methylene group under time interference. Electrostatic adsorption (EA), H-bood (HB), and π-π* contribute 63.5 %, 35.3 %, and 1.2 %, respectively, to the adsorption of oxytetracycline (OTC). The density functional calculation shows that the nucleophilic and electrophilic reactions of OTC are easy to occur near oxygen atoms and nitrogen atoms. The Fukui function proves that the free radical weight of the N atom is 0.096, which is further confirmed the theoretical predictions by liquid chromatography and four-stage electrostatic field orbital trap mass spectrometry. Finally, this study provides a deeper insight into the biochar adsorption OTC.