An analytical method based on high-performance liquid chromatography (HPLC) was established for the determination of ε-polylysine hydrochloride (ε-PLH) in food. Samples were subjected to ultrasonic-assisted extraction twice with 0.3 mol.L-1 sodium dihydrogen phosphate solution (pH value 3.0), degreased with dichloromethane and centrifuged at 8 000 rpm for 5 min. The supernatant was purified using an AL-B solid-phase extraction column and then eluted with 30% (V/V) ammonia solution. Isocratic elution was performed using 0.3 mol.L-1 sodium sulfate solution (pH value 3.0) as the mobile phase and ε-PLH was separated using a hydrophase gel column (Protein SEC-400, 300 mm×8.0 mm, 5 μm). The analyte was identified by a diode array detector (λ=210 nm) and quantification was performed using external standards. The retention time of ε-PLH was 8.2 min. The established method demonstrated good linearity in the range of 5~200 μg.mL-1, with linear correlation coefficients (r2) ≥ 0.99. The limit of detection (LOD) was 15 mg.kg-1 and the limit of quantification (LOQ) was 50 mg.kg-1. The spiked recoveries of this method were investigated in six types of food matrices (jam, rice flour, vinegar, beverages, fried dough products, and mixed grain products). The recoveries of ε-PLH ranged from 90.74% to 106.07% at the three spike levels of 1LOQ, 2LOQ, and maximum residue limit (MRL), respectively, with relative standard deviations ranging from 1.85% to 5.24% (n=6). Overall, this method showed high stability, strong applicability, superior anti-interference ability, good sensitivity, and good reproducibility, thus providing technical support for the monitoring of ε-PLH content in imported and exported food and the formulation of standards.