Background: Shigella dysenteriae is one of the members of Shigella genus which was the main responsible of different Shigellosis outbreaks worldwide. The increasing consumption of antibiotics has led to the emergence and spreading of antibiotic-resistant strains. Therefore, finding new alternatives for infection control is essential, one of which is using bacteriophages. Materials and methods: Lytic bacteriophage against Shigella dysenteriae was isolated from petroleum refinery wastewater. Phage morphological and genetic characteristics were studied using TEM, and sequencing, respectively. In addition, the genome size was estimated, and phage resistance to different temperatures and pH, host range, adsorption rate, and one-step growth were investigated. Results: According to the morphology and genetic results, this phage was named vB-SdyS-ISF003. Sequencing of the PCR products revealed that the vB-SdyS-ISF003 phage belongs to the species T1virus, subfamily Tunavirinae of family Siphoviridae. This was the first detected bacteriophage against S. dysenteriae, which belongs to the family Siphoviridae. In addition, its host range was limited to S. dysenteriae. The genome size was about 62 kb. vBSdyS- ISF003 phage has a number of desirable characteristics including the limited host range to S. dysenteriae, very short connection time, a relatively wide range of temperature tolerance −20 to 50 °C, pH tolerance of 7–9 without significant reduction in the phage titer. Conclusion: vB-SdyS-ISF003 is a novel virulent T1virus phage and has the appropriate potential for being used in bio controlling of S. dysenteriae in different condition.