This study optimised biomass production of health-promoting bacteria by optimising cultivation media using design of experiments methodology. Screening of seven lactic acid bacteria were conducted, where Lacticaseibacillus rhamnosus LRH30 (L. rhamnosus LRH30) was selected as the most suitable candidate for further optimization. Response surface modelling was employed to optimise four dairy-based carbon sources: lactose, whey permeate (WP), demineralised whey (DW) and skim milk powder (SMP), in conjunction with yeast extract and peptone using a central composite design. SMP was identified as the best-suited carbon substrate for high biomass yields, with optimal media concentrations of 12.5 g L-1 lactose, 2.36 g L-1 yeast extract and 2.36 g L-1 peptone in shake flasks and later in batch bioprocess, resulting in biomass quantities of 6.5 g L-1. Additionally, the immunomodulatory effects of L. rhamnosus LRH30 were confirmed in an enzyme-linked immunosorbent assay (ELISA) that showed a significant reduction of tumour necrosis factor (TNF)-α.