Chilled beef is a perishable food product. Due to the broad-spectrum antimicrobial properties of chitosan, and the biodegradability of PLLA, chitosan and PLLA are introduced double bonding groups to obtain Chitosan-graft-Glycidyl methacrylate (Cs-g-GMA) and Poly(L-lactic acid)-co-poly(butylene itaconate) (PLBI), respectively. Cs-g-GMA is then grafted onto the surface of PLBI films by UV curing to produce PLBI/Cs-g-GMA films with non-migratory, broad-spectrum antimicrobial, biodegradable, and possess good oxygen barrier properties, compared to current antimicrobial films. Antimicrobial tests in vitro confirmed that PLBI/Cs-g-GMA film could effectively inhibit the growth and reproduction of P. fluorescens, S. aureus, and E. coli. The PLBI/Cs-g-GMA films were further used for the packaging of chilled beef during 15 days of refrigerated storage. The microbiological (total viable counts (TVC), microbial diversity analysis), physicochemical (pH, total volatile basic nitrogen (TVB-N), drip loss, color) and sensory of beef were measured to evaluate the preservation effect. The results showed that PLBI/Cs-g-GMA films can significantly inhibit Pseudomonas and Aeromonas at the phylum and genus levels, respectively. Based on the good results of the preservation index, PLBI/Cs-g-GMA film could delay the quality deterioration of chilled beef by 3 to 9 days, suggesting that its considerable prospects for maintaining the storage quality of chilled beef.