An appropriate selection of Lactobacillus strain (probiotic/starter/functional) on the basis of its techno-functional characteristics is required before developing a novel fermented functional food. We compared vitamin B-12 (B-12, cobalamin) producing Lactobacillus plantarum isolates, BHM10 and BCF20, for functional (vitamin over-production, genomic insight to B-12 structural genes, and probiotic attributes) and technological [milks (skim and soy) fermentation and B-12 bio-fortification] characteristics. Addition of B-12 precursors (5-amonolevulinate and dimethylbenzimidazole) to cobalamin-free fermentation medium increased vitamin production in BHM10, BCF20, and DSM20016 (a positive standard) by 3.4-, 4.4-, and 3.86-folds, respectively. Three important B-12 structural genes were detected in L. plantarum species (strains BHM10 and BCF20) by PCR for the first time. The gene sequences were submitted to NCBI GenBank and found phylogenetically closer to respective sequences in B-12 producing Lactobacillus reuteri strains. During comparative probiotic testing, BCF20 showed significantly higher (p < 0.05 to p < 0.001) gastrointestinal tolerance and cell surface hydrophobicity (p < 0.05) than BHM10. Moreover, only BCF20 was found positive for BSH activity and also exhibited comparatively better antagonistic potential against potent pathogens. Conversely, high acid and bile susceptible strain BHM10 displayed significantly higher soy milk fermentation and resultant B-12 bio-fortification abilities during technological testing. Two B-12 quantification techniques, UFLC and competitive immunoassay, confirmed the in vitro and in situ bio-production of bio-available form of B-12 after BHM10 fermentation. Conclusively, techno-functional differentiation of two B-12 producing strains elucidates their diverse future use; BCF20 either for B-12 over-production (in vitro) or as a probiotic candidate, while BHM10 for cobalamin bio-fortification (in situ) in soy milk.