The invention belongs to the technical field of nano-drugs, and in particular, relates to an amphotericin B (AmpB) and β-1,3-glucanase (Gls) loaded bi-functional nano-system with both targets, a preparation method and an application thereof. The nano-system provided by the invention comprises Gls and AmpB loaded chitosan nanoparticles. The β-1,3-glucanase modified chitosan nanoparticles are constructed for the first time, which can achieve a bi-functional nano-system both targeting fungal biofilm exopolysaccharide β-1,3-glucan and internal thalli; meanwhile, the β-1,3-glucanase and the amphotericin B have a synergistic interaction effect, so that the removal capacity is improved. The double-loaded nano-system is used for fungal biofilm damage removal for the first time.