Elevated levels of doxycycline (DC) have been detected in the environment due to its extensive utilization as a veterinary antibiotic. Sorption–desorption behavior of DC in soil affects its transport, transformation, and availability in the environment. Thus, sorption–desorption behavior of DC was explored in three soils (S1, S2, and S3) after manure application with and without mesquite wood-waste-derived biochar (BC) pyrolyzed at 600 °C. Sorption batch trials demonstrated the highest DC sorption in soil S1 as compared to S2 and S3, either alone or in combination with manure or manure + BC. Chemical sorption and pore diffusion were involved in DC sorption, as indicated by the kinetic models. Soil S1 with manure + BC exhibited the highest Langmuir model predicted sorption capacity (18.930 mg g⁻¹) compared with the other two soils. DC sorption capacity of soils was increased by 5.0–6.5-fold with the addition of manure, and 10–13-fold with BC application in a soil–manure system. In desorption trials, manure application resulted in 67%, 40%, and 41% increment in DC desorption in soil S1, S2, and S3, respectively, compared to the respective soils without manure application. In contrast, BC application reduced DC desorption by 73%, 66%, and 65%, in S1, S2, and S3, respectively, compared to the soils without any amendment. The highest DC sorption after BC application could be due to H bonding, π–π EDA interactions, and diffusion into the pores of BC. Hence, mesquite wood-waste-derived BC can effectively be used to enhance DC retention in contaminated soil to ensure a sustainable ecosystem.