The methylation (CH3) capacity is dependent on the availability of B vitamins in one carbon metabolism. This study evaluated B vitamin (B9 and B12) deficiency status and its impact on the Diagnostic Accuracy (DA) of DNA methylation among the working population. DNA methylation (5 methylcytosine) and B vitamins were quantified using the ELISA methods. Based on the cut-off values of B vitamins, the subjects were categorized into adequate, marginal, and deficiency status. Fagan’s nomogram was used to assess the DA of DNA methylation in different conditions of B vitamin. DA metrics are used in this study as a Diagnostic Odd Ratio (DOR), a positive Likelihood Ratio (LR+), a negative Likelihood Ratio (LR-), and a posttest probability of true positive DNA methylation that is less than 2.0%. DNA methylation was significantly lower in marginal and deficient B-vitamin status as compared to adequate status. The DA metrics at 5 mC showed high sensitivity and modest specificity for marginal and deficient B vitamins. In vitamin B9 deficiency, LR (+) was 2.51 (95% CI: 0.99–6.39), DOR was 10.4 (95% CI: 2.06–51.2), and the post-test probability was 92% (95% CI: 83–97). In vitamin B12 deficiency, LR (+) was 1.58 (95% CI: 1.03–2.44), DOR was 3.16 (95% CI: 1.29–7.66), and the post-test probability was 86% (95% CI: 80–90). In B vitamin deficiency, DA metrics of DNA methylation: LR+, LR-, DOR, and post-test probability were more precisely predicted. DNA hypomethylation could be used as a predictive biomarker in B vitamin deficiency. The findings of the study help to prevent adverse health effects due to methylation capacity loss.