Amphotericin B is a macrolide antibiotic that can selectively bind to fungal cell membranes, leading to the leakage of intracellular substances and death. However, due to its high toxicity and difficulty in crossing the blood-brain barrier, improving encapsulation rate and reducing toxicity are necessary conditions for enhancing therapeutic efficacy. To control the release rate of the drug, reduce peak and trough levels in the blood, and lower systemic drug levels, biodegradable materials can be used as drug carriers. The present invention proposes an amphotericin B sustained-release microsphere and its preparation method. The drug carrier is a biodegradable polymer, resulting in sustained-release microspheres with a particle size of less than 120nm, stable release for over 80 hours, and a polymer weight average molecular weight of 5000-50000. The preparation method involves: the oil phase consists of a biodegradable polymer and amphotericin B dissolved in acetone solution with a cosolvent, and the water phase contains distilled water to which an emulsifier has been added. A solution of physiological saline or 5% glucose injection is prepared by adding the oil phase to the water phase, stirring, allowing natural evaporation to remove acetone, dialyzing, and then lyophilizing into a powder to obtain the desired medication.