Abstract: To prepare microgels laden with ε-polylysine (PL) in this investigation, chitosan (CS) and bacterial cellulose (Bacterial Cellulose, BC) were exploited as substrates, and a simplistic and gentle preparation procedure was implemented. The key process parameters of the microgels (PL-BC/CSM) were ascertained, and their microscopic morphology, structural traits, thermal stability, antibacterial activity, and sustained-release properties were characterized. The findings suggested that under the preparation circumstances of a BC/CS composite volume ratio of 10:90, an ST addition of 2%, a PL content of 0.4 mg/mL, a homogenization velocity of 10000 r/min, and a duration of 2 min, microgels with an encapsulation efficiency of 48.23%, a particle size of 1423 nm, a polydispersity index (PDI) of 0.25, and a Zeta potential of 33.26 mV could be attained. In comparison with the physical composite group lacking a crosslinking agent (BC/CSM), the outcomes of SEM and TEM manifested that the PL-BC/CSM group exhibited an outstanding surface morphological structure and thermal stability. FTIR analysis revealed that PL molecules were successfully incorporated into the polymer network. The establishment of PL-BC/CSM could facilitate the sustained release of PL, which was conducive to enhancing its utilization rate. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against S. aureus were 26 and 52 μg/mL, respectively, and those against E. coli were 52 and 104 μg/mL, respectively, demonstrating satisfactory antibacterial efficacy. This study provides a theoretical foundation for the application of such microgels in the domain of food.