Microneedles with insulin-loaded glucose-responsive particles are promising to control the blood glucose levels of diabetic patients. In particular, the long-term usage of these microneedles calls for biodegradable and cost-effective particles, which are still large challenges. In this paper, glucose-responsive 4-carboxy-3-fluorophenylboronic acid-grafted ε-polylysine (CFPBA-g-PL) was synthesized to meet these requirements. CFPBA-g-PL had low cytotoxicity, good hemocompatibility and no tissue reaction. The pharmacokinetics of CFPBA-g-PL were also studied. The self-assembled particles of CFPBA-g-PL were prepared via simple ultrasonic treatment. The insulin-loaded particles of CFPBA-g-PL (named INS/GRP-12.8) presented a glucose-responsive insulin delivery performance based on the disassembly-related mechanism in vitro. The INS/GRP-12.8-encapsulated microneedle patch with a uniform morphology and moderate skin penetration performance was prepared via a molding strategy. INS/GRP-12.8 lasted for more than 8 hours of normoglycemia on STZ-induced diabetic SD rats via subcutaneous injection and the INS/GRP-12.8-encapsulated microneedle patch also showed a blood-glucose-level-lowering performance in vivo via transdermal administration.