Research on the efficient flotation desilication of low-grade magnesite is of great significance for the sustainable development of magnesium resources. Traditional collectors usually have some disadvantages, such as poor selectivity, severe environmental pollution, and weak water solubility. To strengthen the desilication flotation process of magnesite ore, the biodegradable surfactant, cocamidopropyl amine oxide (CPAO), was first utilized as the collector for the separation of the magnesite and quartz. The selective adsorption behavior and mechanism of the quartz and magnesite with the CPAO as the collector were studied through the micro-flotation experiments of the single mineral and the artificially mixed mineral, contact angle and atomic force microscopy (AFM) measurements, fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analyses. The flotation results indicated that the CPAO showed good selectivity and could effectively separate magnesite and quartz. When the concentration of the CPAO was 10.0 mg/L in the natural pulp pH (about 7.2), the concentrates with 97.67% MgO recovery and 45.62% MgO grade were obtained. The contact angle and AFM measurements indicated that the CPAO could selectively adsorb on the quartz surface rather than the magnesite surface to improve the interface difference between them, especially its surface hydrophobicity. The results of the FTIR and XPS analyses indicated that the CPAO is selectively adsorbed on the surface of the quartz, mainly through electrostatic interaction and hydrogen bonding. In conclusion, the CPAO had good selectivity and great potential as an effective collector in the reverse flotation desilication progress of magnesite.