Mirsayafi was sentenced to two years in prison for allegedly insulting Iran's leaders.
Prison officials have told Mirsayafi's family that he overdosed on sedatives, but the family has said that the circumstances of his death remain suspicious.
French media watchdog Reporters Without Borders has called for an independent inquiry into the blogger's death.
In the open letter, the bloggers write that there is nothing offensive or insulting about Mirsayafi's blog entries. They ask Khamenei directly to explain which of Mirsayafi's writings were insulting and landed him in prison.
They also ask why Mirsayafi's trial was held behind closed doors and why he didn't have the right to defend himself publicly.
The bloggers also noted that Khamenei had said in a speech last year that there is freedom to criticize the leader. How then, they ask, should they criticize Khamenei without ending up in jail?
-- Golnaz Esfandiari