Amphotericin B (AmB) is a large amphiphilic molecule, which is a drug of choice in therapy of systemic fungal infection because of its board-spectrum antifungal activity. However, conventional formulation of AmB is micelles, which is unstable in the bloodstream and releases drug to form toxic aggregates. Lipid complex of AmB has been developed to reduce toxicity while retaining activity. Microfluidic method is mixing technique, which permits millisecond mixing at the nano/microliter scale, can reproducibly generate limit size of particles of drug delivery system. In this study, microfluidic hydrodynamic focusing method was used to produce AmB lipid complex. It was obtained of two synthetic phospholipids: hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylglycerol (DSPG) and AmB in the molar ratio of 1:1:2.The lipid complex of AmB was characterized using differential scanning calorimetry (DSC), X -ray diffraction, Fourier-transform infrared spectroscopy (FTIR) and Field Emission Scanning Electron Microscopy(FESEM).The results showed that AmB lipid complex, prepared by microfluidic method, had smallest size at the flow rate ratio (FRR) of 5:1. The distinct phase transition temperature, the crystal peak of phospholipid, the characteristic band of-P-O-group of phospholipid were not observed in the DSC curve, X-ray diffraction diagram and FTIR spectra, respectively, of AmB lipid complex.