Despite the excellent antimicrobial activity, the high toxicity and low selectivity of cationic antimicrobial peptides (AMPs) and their synthetic analogues impede their biomedical applications. In this study, we report a series of cationic peptidopolysaccharides synthesized by thiol-ene click chemistry of grafting antimicrobial polypeptides, methacrylate-ended poly(lysine- random-phenylalanine) (Me-K nF m), onto a thiolated polysaccharide (dextran, Dex) backbone. Their copolymers (Dex- g-K nF m) exhibit potent broad-spectrum antibacterial and antifungal activity against Gram-negative bacteria ( Pseudomonas aeruginosa and Escherichia coli), Gram-positive bacteria [methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis], and fungi ( Candida albicans) with minimal inhibitory concentrations in the range of 31.25-500 μg·mL-1. More importantly, Dex- g-K nF m copolymers did not induce drug resistance of MRSA up to 17 passages. In addition, these copolymers have an improved hemocompatibility and exhibit good in vitro biocompatibility with murine myoblast (C2C12) cells. Among the synthesized peptidopolysaccharides, DexL- g-K12.5F12.5-50%, as the optimal agent, displayed a selectivity more than 200 times the maximum value of polypeptide molecules. Furthermore, a strong in vivo antimicrobial efficacy with a log reduction above 3 in a mouse bacterial sepsis model has been obtained. These excellent biological properties present a promising prospect for Dex- g-K nF m in biomedical applications.