By increasing the environmental pollution, crop losses, and side effects of chemically synthesized vitamins; new vitamin sources should be included. Through this study, we introduce novel riboflavin bacterial producer Novosphingobium panipatense-SR3 (MT002778) and tested various nutritional factors with interactions effects on the production abilities. Yeast extract, maltose, and glycine were the best nitrogen, carbon, and amino acid sources for enhancing the production, respectively. The interaction between the previous factors with three concentrations of each (+, 0, −) studied statistically using Box–Behnken statistical quadric design 13- run. The perfect interaction increases the production to 497.12 mg/l (predicted 489.45 mg/l) using 30 g/l maltose, 10 g/l yeast extract, and 1 g/l glycine. The F and P- values of the tested model of riboflavin and OD600 indicating significant results with probability ≤ 0.05. Also, the evaluating statistical parameter coefficient (R²) was 0.994 of riboflavin and 0.992 of OD600 with adjusted R² value 0.976, and 0.967, respectively, which indicated that the whole variations were explained highly by the statistical model. The novel producer proved its high riboflavin production ability especially under the optimized conditions comparing with previous producers and represents a new high-speed riboflavin producer that could utilize in the industrial process.