Snake venoms are mostly composed of various proteins and peptides with toxicity and pharmacological effects depending on their geographical sources. Naja naja oxiana is one of the most medically important venomous snakes in Iran and Central Asia. The bite of this type of snake can cause severe pain and swelling, as well as neurotoxicity. Without medical treatment, symptoms quickly worsen and death can occur soon. A detailed understanding of venom components can provide new insight into the production of antivenom against toxic agents instead of crude venom. Specific antibodies against toxic fractions are of utmost importance in neutralizing crude venom. Therefore, the proteome profile of these fractions of Naja naja oxidana venom was analyzed using fractionation by gel filtration, two-dimensional electrophoresis, mass spectrometry, and data mining. Base on the results, in total, 32 spots were detected and categorized into three protein families, namely three-finger toxin (3FTx), phospholipase, and Cysteine-rich secretory proteins (CRISP). These proteins consist of more than 70% crude venom all with a molecular weight below 25 kDa. The 3FTx as a highly diverse constituent in the venom of Naja species was in large quantity in this district. Short-chain neurotoxins, including short neurotoxin, cytotoxin, and muscarinic toxin-like protein, were in abundance, respectively. In conclusion, the recognition of toxic fractions of Naja naja oxiana in this region could be of great help in the production of an effective antivenom against similar compositions. It can also help the medical care department to find out the clinical sign of cobra venom. To the best of our knowledge, this was the first study to report the proteomic of toxic fractions of Naja naja oxiana in Iran.