To identify optimal growth substrates for enhancing fruit quality and antioxidant capacity of southern highbush blueberry cultivars, this study evaluated substrate formulations using coconut coir and peat moss. Two-years-old ‘Eureka’ and ‘Misty’ plants were cultivated in four coconut coir substrates under controlled greenhouse conditions. Blueberry fruit quality, skin coloration, biochemical composition, and antioxidant enzyme activity were analyzed. Data were subjected to ANOVA and membership function analysis for comprehensive evaluation. The results indicated that for the ‘Eureka’ blueberry, the T1 (100% coconut coir) substrate showed significant advantages in terms of fruit longitudinal diameter, firmness, TSS content, anthocyanin content, total phenolic content, total flavonoid content, and SOD activity. For the ‘Misty’ blueberry, the T4 (peat soil: coconut coir=1:1) substrate demonstrated significant advantages in terms of fruit anthocyanin content, total phenolic content, and total flavonoid content. It could be concluded that different blueberry varieties had their own optimal coconut coir content, with T1 and T4 being the substrates that yield the best fruit quality and strongest antioxidant capacity for ‘Eureka’ and ‘Misty’, respectively.