Salinity and drought are key abiotic stresses limiting Persian walnut (Juglans regia) productivity worldwide, particularly in arid and semi-arid regions. To address these challenges and enhance stress tolerance, somatic embryos of J. regia cv. Chandler were co-transformed with flavodoxin (Fld) and betaine aldehyde dehydrogenase (BADH) genes using Agrobacterium tumefaciens harboring the binary vector pBI121. Putative transgenic embryos were selected on antibiotic media and directed to germination and plantlet regeneration. Polymerase chain reaction (PCR) analysis confirmed that transgene had successfully integration into the genomes of four lines 4, 6, 10, and 12 with a transformation efficiency of 4.44%. Also, the expression of both target genes was confirmed using reverse transcription PCR (RT-PCR) in the four lines. Transgenic plants (Line 4, 6, 10, and 12) along with wild-types (WT) controls were subjected to salinity (0, 50, 100 and 200 mM NaCl) and osmotic stress using polyethylene glycol (PEG) at concentrations of 0%, 5%, 10% and 15% for 30 days. Compared to controls, transgenic lines exhibited a noticeably higher survival rate, more healthy leaves, higher plant height, and higher levels of proline and glycine betaine (GB). Among the transgenic lines tested, Line 4 exhibited the highest stress tolerance. Although glycine betaine levels were comparable among lines under certain conditions, Line 4 showed superior physiological performance, likely due to the combined effects of BADH and enhanced Fld gene activity, which improved photosynthetic efficiency under stress. Overall, co-transformation of Fld and BADH genes conferred enhanced photosynthetic capacity and osmotic regulation, thereby improving the overall abiotic stress tolerance of transgenic Persian walnut. Key message Co-transformation of Fld and BADH genes enhanced significantly salinity and drought tolerance in transgenic Persian walnut plantlets.