The 37-year-old man and 34-year-old woman are being held over the deaths of two boys, aged 13 and 14, who were found seriously ill in a house in Gregg House Road, Shiregreen, this morning.
They were taken to hospital, where they later died.
Four other children – aged 11, 10, three and a baby of seven months – were also taken to hospital, where they remain this afternoon.
Emergency services were called to the property at 7.30am and there were multiple police cars and ambulances deployed.
An air ambulance was also scrambled.
The house where the children were found is cordoned off this afternoon and crime scene investigators in protective white suits are examining the property.
The cause death for both boys has not yet been established.
Post mortem examinations are planned.
Detective Chief Inspector Simon Palmer said: “Detectives and specialist officers remain in the Gregg House Road area carrying out enquiries and we are working hard to ascertain the exact circumstances surrounding this incident.
“We will remain in the area this afternoon and into the evening carrying out enquiries and providing reassurance.
“I appreciate the events of this morning have caused concern locally but I’d like to reassure you that there is no wider risk to the community. We will be providing regular updates when we are able to. If anyone does have concerns, please do not hesitate to speak to an officer in the area.”
Anyone with concerns or information should call South Yorkshire Police on 101 or Crimestoppers, anonymously, on 0800 555111 quoting incident number 122 of May 24.