Objective To establish a method for determining the total vitamin B₁₂ content in infant formula milk powder based on the indirect detection of α-ribazole, a degradation product and nucleoside component of vitamin B₁₂.Methods After protein denaturation and sugar removal, the sample underwent acid hydrolysis and enzymatic dephosphorylation to release α-ribazole, which was then purified using a boronate affinity column. Quantitative analysis was performed using hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD).Results Alpha-ribazole showed good linearity in the range of 1.00~10.0 μg/L, with a correlation coefficient (R2) of 0.999 1. The spiked recovery ranged from 72.5% to 78.3%, with relative standard deviations (RSDs) between 4.56% and 7.28%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.4 μg/100 g and 1.2 μg/100 g, respectively. Bland-Altman analysis showed no significant difference between this method and the national standard method, and the consistency was good.Conclusion This method is highly sensitive and reproducible, and it can accurately determine the total vitamin B₁₂ content in infant formula milk powder without the need for cyanide pretreatment.