The present work is focused on design and development of novel Amphotericin B-conjugated biocompatible and biodegradable poly-peptide hydrogels to improve the antifungal activity. Using three kinds of promoting self-assembly groups (2-naphthalene acetic acid, Naproxen and Dexamethasone) and poly-peptide sequence (Phe-Phe-Asp-Lys-Tyr, FFDKY), we successfully synthesized the Nap-FFDK (AmB) Y gels, Npx-FFDK (AmB) Y gels and Dex-FFDK (AmB) Y gels. The Amphotericin B-conjugated hydrogelators are highly soluble in different aqueous solutions. The Cryo-transmission electron microscopy (TEM) and scanning electron microscopy (SEM) micrographs of hydrogels afford nanofibers with width of 20~50 nm. Powder X-ray diffraction (PXRD) analyses demonstrate that the crystalline structure of the AmB and Dex are changed into amorphous structure after formation of hydrogels. Circular dichroism (CD) spectra of the solution of blank carriers and the corresponding drug deliveries further help elucidate the molecular arrangement in gel phase, indicating the existence of turn features. The in vitro drug releases suggest that the Amphotericin B-conjugated hydrogels are suitable as drug-controlled release vehicles for hydrophobic drug. The antifungal effect of AmB-conjugated hydrogels significantly exhibits the antifungal activity against Candida albicans. The results of present study indicated that the Amphotericin B-conjugated hydrogels are suitable carriers for poorly water soluble drugs and for enhancement of therapeutic efficacy of antifungal drugs.