Abstract: Invasive fungal infections are spreading around the world, causing over 1.5 million deaths worldwide each year. Potent antifungal agents with low toxicity are urgently needed due to the limited antifungal drugs available. Host defense peptides (HDPs) demonstrated potent activity against microorganisms because of their broad-spectrum antimicrobial activity and insusceptibility to antimicrobial resistance. However, the wide application of HDPs is hindered by their inherent shortcomings such as difficult synthesis, expensiveness and poor proteolytic stability. To resolve these problems, a series of β-amino acid polymers (DAP_xPh_y)₂₀ were designed and synthesized by mimicking the structure of HDPs. The random copolymers were obtained by controllable and water insensitive ring-opening polymerization of β-amino acid N-thiocarboxyanhydrides (β-NTA) with N(α)-Z-DL-2,3-diaminopropionic acid N-thiocarboxyanhydrides (DAP) as the cationic monomer and 3-amino-2-(phenyl) propionic acid N-thiocarboxyanhydrides (Ph) as the hydrophobic monomer. The chain length and structure of polymers were characterized by ¹H-NMR and gel penetration chromatography (GPC). Results showed that (DAP_xPh_y)₂₀ had potent antifungal activity to C. albicans K1 with minimum inhibitory concentration (MIC) at 1.56—12.5 μg/mL. The antifungal activity weakened with the increasing of Ph content, since the reduction of the positive charge ratio might lead to a weakened electrostatic interaction with the fungal cell membrane. These β-amino acid polymers did not cause significant hemolysis of human red blood cells at the mass concentration of 400 μg/mL and also exhibited low cytotoxicity upon NIH 3T3 fibroblast cells at 200 μg/mL. This study demonstrated that (DAP_xPh_y)₂₀ exhibited potent antifungal activity with low hemolysis, cytotoxicity and easy preparation, showing broad potential in the treatment of fungal infections.