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Riboflavin (Vitamin B₂) is an essential vitamin and a microbial metabolite produced by some lactic acid bacteria (LAB). This investigation aims to study the overproduction of riboflavin in selected <i>Lactiplantibacillus plantarum</i> strain by using the one factor at a time (OFAT) tool coupled with the Fuzzy Inference System (FIS) and its validation through fermentative production in semi-defined media. Out of three <i>Lactiplantibacillus</i> strains used in this study, the maximum riboflavin producing strain was selected based on its ability to grow and produce higher levels of riboflavin. In results, <i>Lactiplantibacillus plantarum</i> strain MTCC 25432 was able to produce 346 µg/L riboflavin in riboflavin deficient assay medium and was investigated further. By using the OFAT–fuzzy FIS system, casamino acid in the range of 5–20 g/L, GTP 0.01–0.04 g/L, sodium acetate 5–15 g/L, and glycine 5–15 g/L were used to predict their effect on riboflavin production. The conditions optimized with modeling showed a 24% increment in riboflavin production (429 µg/L) by <i>Lactiplantibacillus plantarum</i> MTCC 25432 vis-a-vis the unoptimized counterpart (346 µg/L). In conclusion, an FIS-based predictive model was effectively implemented to estimate the riboflavin within an acceptable limit of 3.4%. Riboflavin production enhancing effects observed with various levels of sodium acetate, casamino acid, and GTP could be useful to re-design matrices for riboflavin production.