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Hydrogels have emerged as promising biomaterials due to their excellent performance; however, their biocompatibility, biodegradability, and absorbability still require improvement to support a broader range of medical applications. This paper presents a new biofunctionalized hydrogel based on in situ crosslinking between maleimide-terminated four-arm-poly(ethylene glycol) (4-arm-PEG-Mal) and poly(ε-lysine) (ε-PL). The PEG/ε-PL hydrogels, named LG-n, were rapidly formed via amine/maleimide reaction by mixing 4-arm-PEG-Mal and ε-PL under physiological conditions. The corresponding dry gels (DLG-n) were obtained through a freeze-drying technique. 1 H NMR, FT-IR, and SEM were utilized to confirm the structures of 4-arm-PEG-Mal and LG-n (or DLG-n), and the effects of solid content on the physicochemical properties of the hydrogels were investigated. Although high solid content could increase the swelling ratio, all LG-n samples exhibited a low equilibrium swelling ratio of less than 30%. LG-7, which contained moderate solid content, exhibited optimal compression properties characterized by a compressive fracture strength of 45.2 kPa and a deformation of 69.5%. Compression cycle tests revealed that LG-n demonstrated good anti-fatigue performance. In vitro degradation studies confirmed the biodegradability of LG-n, with the degradation rate primarily governing the drug (ceftibuten) release efficiency, leading to a sustained release duration of four weeks. Cytotoxicity tests, cell survival morphology observation, live/dead assays, and hemolysis tests indicated that LG-n exhibited excellent cytocompatibility and low hemolysis rates (<5%). Furthermore, the broad-spectrum antibacterial activity of LG-n was verified by an inhibition zone method. In conclusion, the developed LG-n hydrogels hold promising applications in the medical field, particularly as drug sustained-release carriers and wound dressings.