Hyaluronic acid (HA), composed of glucuronic acid (GlcUA) and N-acetyl glucoseamine (GlcNAc), is a versatile biopolymer with high commercial value and innumerous physiological roles and pharmaceutical applications. ThehasA gene has main role in HA biosynthesis byStreptococcusstrain as a natural producer. ThehasB andhasC genes are also mediate GlcUA precursor biosynthesis. In the present study,S. equisimilis hasA gene;B. subtilis tuaD andgtaB genes for GlcUA precursors enhancement, andvgbgene coding bacterial hemoglobin as an oxygen provider were used to construct theB.subtilisstrain for HA production. RBSHA (hasA), RBSHA2 (hasA/tuaD/gtaB), and RBSHA3 (hasA/tuaD/gtaB/vgb) strains were developed and confirmed through genotype and phenotype analysis. After HA production and purification, FTIR spectroscopy confirmed the produced HA structures. HA assay showed the highest HA titer for RBSHA3 (2.1 ± 0.18 mg/ml) and then RBSHA2 (1.9 ± 0.03 mg/ml), and RBSHA (0.6 ± 0.14 mg/ml). Statistical analysis indicated there is no significant difference in HA titer between RBSHA2 and RBSHA3 strains (p-value > 0.05), however, these strains produced HA approximately 4-fold higher than that of RBSHA strain. Agarose gel electrophoresis showed the same molecular weight (< 30 kDa) of produced HA by strains. Dynamic light scattering (DLS) revealed all HA polymers had a relatively low polydispersity index (PDI < 0.5). These findings demonstrate the successful GlcUA biosynthetic pathway engineering strategy in improving HA yield by recombinantB. subtilis, metabolically-robust, and industrially potential strain.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13568-023-01567-2.