The azithromycin-rhamnolipid (AZI-RHL) micelles were prepared by thin film hydration method. Taking encapsulation efficiency and drug loading as evaluation indicators, the optimal preparation technology was obtained through single factor test and orthogonal test, and then the physical and chemical properties of AZI-RHL micelles were investigated. The critical micelle concentration (CMC) of RHL aqueous solution was determined before preparing the AZI-RHL micelles. The results showed that the CMC of RHL aqueous solution was about 0.25 mg/mL. The optimized preparation technology conditions were as follows: RHL dosage was 100 mg, methanol dosage was 12 mL, and the stirring time was 20 min. The AZI-RHL micelles prepared under the optimized preparation technology conditions were spherical when observed under transmission electron microscope (TEM), with a hydrodynamic diameter of 136.3±68.5 nm, a Zeta potential of -23.1±6.8 mV, an encapsulation efficiency of 80.34%±0.60%, and a drug loading of 19.42%±0.48%. Infrared spectroscopy revealed that AZI was embedded in micelles. The in vitro cumulative release test showed that AZI-RHL micelles had a certain sustained release effect compared with bulk drugs. The cumulative release curve of AZI-RHL micelles in vitro conformed to the Ritger-Peppas equation, and the release of drugs was dominated by Fick diffusion. To sum up the above, the preparation technology of AZI-RHL micelles was stable and reliable; the AZI-RHL micelles had small particle size, high encapsulation efficiency and high drug loading. AZI-RHL micelles achieved synergistic delivery of RHL and AZI by means of formulations. RHL had good anti-biofilm properties, and in combination with AZI, AZI-RHL micelles can exhibit greater anti-biofilm potential. In addition, rhamnolipids, as small-molecule biosurfactants, had the characteristics of low toxicity and easy degradation compared with other types of surfactants. AZI-RHL micelles enriched the research on surfactant micelles as drug carriers, and provided a certain reference for the preparation of subsequent surfactant micelle drug delivery systems.