In this study, the optimal conditions and effects of external factors on tetracycline adsorption by magnetic multi-walled carbon nanotubes (MMWCNTs) were established by a response surface methodology for the first time. Batch adsorption experiments showed that increasing the dosage and contact time effectively promoted the adsorption of tetracycline and maximum removal of 97.93–99.13% was achieved at pH 3–7. The pseudo-second-order model and Fourier-transform infrared spectroscopy spectra indicated that the mechanism of adsorption may be π–π electron interaction and cation–π electron bonding. Design Expert was utilized to develop a response surface methodology for the analysis and optimization of tetracycline adsorption by magnetic multi-walled carbon nanotubes. The Box–Behnken design (BBD) results showed that the optimization exhibited high significance and reliability. The main effect plots and Pareto chart indicated that pH exerted a significant individual effect on the regulation of adsorption, while 3D response surface plots and interaction effect plots exhibited a significant antagonistic interaction between pH and contact time. A maximum tetracycline removal of 99.16% was achieved under the optimal conditions of 12 mg adsorbent dosage at pH 5.43, with an adsorption time of 120 min. Mathematical and experimental results confirmed the accuracy of the established optimal conditions.