Two strains were selected i.e Lactobacillus acidophilus NB1MCC1380, Lactobacillus bulagricus L. B1449 among  Lactococcus lactis NRRL B 23802 and Streptococcus thermophilus NRC2010 to obtain  as a result of protoplasting fusion.  the latter strains were known as a good folic acid producer. In this technique polyethylene glycol 1000 and lysozyme enzyme for induction of protoplasting process which was more efficient for time period of 24, 48 hours from protoplasting  imitating. All obtained recombinant  fusant have been evaluated for folic acid production by using high performance liquid chromatography HPLC. The results showed that the two parameter the retention time of folic acid was 3.7 min and the concentration of folic acid in the fermentation  media ranged  between (25.7 µg\ L to 42.9µg\L ) in the parental strain Lactococcus lactis while it was 42.9µg/L. In Streptococcus thermophilus, The fusant strains produced the following concentration of folate: Fusant 1 (32.67 µg\L), fusant 2 (25.7 µg\L), fusant 3 ( 26.04 µg\L), fusant 4 (25.9 µg\ L) respectively.