The demand for innovative plant-based probiotic beverages is growing rapidly. This study aimed to develop and evaluate a novel kefir beverage using two types of sesame milk: permeate-based sesame milk (PSM) and water-based sesame milk (WSM). Chemical, physical, microbial, and sensory properties of kefir were assessed. The total solids content (protein, fat, carbohydrates, and ash) in fresh kefir was 12.68, 13.31, and 16.38% for cow's milk kefir (CMK), WSM kefir (WSMK), and PSM kefir(PSMK), respectively, andincreasedslightlyafter14daysof storage, reaching 13.18, 13.53, and 16.56%. The fresh PSMK exhibited notable mineral content, containing (mg/100g) 258.23 Ca, 137.14 P,70.24K, and smaller amounts of Na, Mg, Cu, Fe, Zn, and Mn, along with 5.18μg/100g of Se. In terms of volatile compounds, PSMK had the highest acetaldehyde concentration (7.48 mg/L), followed by CMK (4.91mg/L) and WSMK (4.44mg/L). Ethanol levels were the highest in fresh WSMK (0.129%). The viscosity and color attributes of PSMK were closely aligned with those of CMK, with the viscosity increasing over time to 1.53, 1.40, and 1.57 cP for PSMK, WSMK, and CMK, respectively. All kefir types supported viable probiotic populations, with PSMK demonstrating superior Lactobacillus and Lactococcus growth compared to WSMK. Sensory evaluations revealed high consumer acceptability for PSMK, comparable to CMK, with a purchase recommendation rate exceeding 76% for both PSMK and WSMK. Scanning electron microscopy revealed that the microstructure of PSMK grains was well-balanced and similar to that of CMK grains. This study highlights PSM as a promising dairy alternative for producing high-quality probiotic kefir, offering consumers an appealing, nutritious option within the growing plant-based beverage market.