Abstract: The aim of this study was to develop a fermented food rich in β-nicotinamide mononucleotide (β-NMN). The influence of fermentation strain, fermentation time, and addition of B-group vitamins on the β-NMN content of fermented Coix (Coix lacryma-jobi) seed was investigated. Based on the results of single-factor experiments, a standardized experimental design was subsequently adopted to optimize the amount of B-group vitamins added, and to evaluate the quality and bioactivity of the fermented Coix seed. The results showed that Rhizopus oryzae was the optimal fermentation strain and the optimal fermentation time was 54 h. The optimal addition amounts of the various B-group vitamins were as follows: nicotinamide: 150 mg/kg, niacin: 50 mg/kg, vitamin B1: 50 μg/kg. Under these conditions, the fermented Coix seed exhibited good flavor and a β-NMN content of up to 20.07 μg/g. It also contained a reducing sugar content of 19.6 g/100 g, organic acid content of 0.37 g/kg, and alcohol content of 2.5%. The contents of γ-aminobutyric acid, S-adenosylmethionine, and total phenols increased by 1.24, 256, and 4.7 times, respectively. The ABTS⁺· scavenging capacity, FRAP, ·OH scavenging capacity and DPPH· scavenging capacity increased by 14.37, 2.40, 4.55, and 4.67 times, respectively. The study provides a novel approach and valuable reference for future research on β-NMN-rich Coix functional foods.