Using agro-industrial by-products as a substrate for the growth of probiotic strains is an alternative for increasing their added value. This study aimed to design and optimize the production of a functional ingredient (FI) with Lacticaseibacillus casei (ATCC 393) immobilized in oat bran (OB). A Box–Behnken experimental design was applied to study the effect of hydration level (HL), whey supplementation, and initial inoculum concentration (IC) on the probiotic growing. According to design, systems containing 1 g of OB, supplemented with 0 to 0.4 g whey/g OB, and HL from 5 to 20 mL of water/g OB were sterilized and inoculated with an IC in the range 5.61 and 7.61 log (CFU/g OB). After incubation at 37°C for 24 h with orbital shaking, the systems were washed, centrifuged, and vacuum-dried. Independent variables were optimized to maximize cell count and growth and minimize cell decay due to drying. The optimal conditions found were for HL: 15 mL of water/g OB; for whey: 0.3 g/g OB, and for the IC: 5.79 log(CFU/g OB), which were recorded within the studied range. This result demonstrates the feasibility of optimizing the production of a dried FI based on OB as a substrate for L. casei.